Compositions comprising bacterially derived minicells and methods of using the same

ABSTRACT

Compositions and methods for treating cancer are provided. In particular, the compositions comprise an anti-neoplastic agent and either an interferon type I agonist or an interferon type II agonist, or a combination of an interferon type I agonist and an interferon type II agonist.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefits under 35 USC § 119 to U.S.provisional Application 62/702,172, filed Jul. 23, 2018, and U.S.provisional Application 62/788,265, filed Jan. 4, 2019, the entirecontents of which are incorporated herein by reference in theirentirety.

BACKGROUND

Currently, most drugs used for treating cancer are administeredsystemically. Although systemic delivery of cytotoxic anticancer drugsplays a crucial role in cancer therapeutics, it also engenders seriousproblems. For instance, systemic exposure of normal tissues/organs tothe administered drug can cause severe toxicity. This is exacerbated bythe fact that systemically delivered cancer chemotherapy drugs oftenmust be delivered at very high dosages to overcome poor bioavailabilityof the drugs and the large volume of distribution within a patient.Also, systemic drug administration can be invasive, as it often requiresthe use of a secured catheter in a major blood vessel. Because systemicdrug administration often requires the use of veins, either peripheralor central, it can cause local complications such as phlebitis.Extravasation of a drug also can lead to vesicant/tissue damage at thelocal site of administration, such as is commonly seen uponadministration of vinca alkaloids and anthracyclines.

Another challenge in cancer therapy is intrinsic or acquired clinicaltumor resistance to chemotherapy. Intrinsic resistance exists at thetime of diagnosis in tumors that fail to respond to first-linechemotherapy. Acquired resistance occurs in tumors that may respond wellto initial treatment, but exhibit a resistant phenotype upon recurrence.Such tumors gain resistance both to previously used drugs and to newdrugs, including drugs with different structures and mechanisms ofaction. The term MDR (multidrug resistance) describes this phenomenon inwhich tumor cells become cross-resistant to several structurallyunrelated drugs after exposure to a single drug. The mechanisms formulti-drug resistance are complex and multifactorial, owing largely tothe high level of genomic instability and mutations in cancer cells.Exemplary mechanisms are drug inactivation, extrusion of drug by cellmembrane pumps, decreased drug influx, mutations of drug targets andfailure to initiate apoptosis. Bredel, 2001; Chen et al., 2001; Sun etal., 2001; and White & McCubrey, 2001.

Interactions between the immune system and malignant cells also play animportant role in tumorigenesis. Failure of the immune system to detectand reject transformed cells may lead to cancer development. Tumors usemultiple mechanisms to escape from immune-mediated rejection. Many ofthese mechanisms are now known on a cellular and molecular level.Despite this knowledge, cancer immunotherapy is still not an establishedtreatment in the clinic.

Accordingly, there remains a great need for delivery systems that canprovide targeted delivery of drugs that can reduce drug resistance,promote apoptosis, and induce immune responses while avoiding theproblems associated with delivering these drugs systemically. Thepresent invention satisfies these needs.

SUMMARY OF THE INVENTION

One embodiment of the invention relates to a composition comprising (a)a therapeutically effective dose of purified, intact bacterially derivedminicells comprising an anti-neoplastic agent, and (b) an interferontype I agonist, an interferon type II agonist, or a combination of aninterferon type I agonist and an interferon type II agonist. Theinterferon type I agonist and/or the interferon type II agonist can beoptionally present within intact bacterially derived minicells.

In one embodiment, the composition comprises (a) a therapeuticallyeffective dose of purified, intact bacterially derived minicellscomprising an anti-neoplastic agent, and (b) a therapeutically effectivedose of purified, intact bacterially derived minicells comprising aninterferon type I agonist. In another embodiment, the compositioncomprises (a) a therapeutically effective dose of purified, intactbacterially derived minicells comprising an anti-neoplastic agent, and(b) a therapeutically effective dose of purified, intact bacteriallyderived minicells comprising an interferon type II agonist. In yet afurther embodiment, the composition comprises (a) a therapeuticallyeffective dose of purified, intact bacterially derived minicellscomprising an anti-neoplastic agent; (b) a therapeutically effectivedose of purified, intact bacterially derived minicells comprising aninterferon type I agonist; and (c) a therapeutically effective dose ofpurified, intact bacterially derived minicells comprising an interferontype II agonist.

In one embodiment, the anti-neoplastic agent and the interferon type Iagonist, the interferon type II agonist, or the combination of aninterferon type I agonist and an interferon type II agonist, arepackaged within two or more purified, intact bacterially derivedminicells. In one embodiment, the anti-neoplastic agent and theinterferon type I agonist, the interferon type II agonist, or thecombination of an interferon type I agonist and an interferon type IIagonist are packaged within three separate populations of purified,intact bacterially derived minicells.

In one embodiment, the composition comprises the anti-neoplastic agent,the interferon type I agonist, and the interferon type II agonist,wherein: (a) the anti-neoplastic agent, the interferon type I agonist,and the interferon type II agonist are comprised within the sameminicell; (b) the anti-neoplastic agent and the interferon type Iagonist are comprised within a first minicell, and the interferon typeII agonist is comprised within a second minicell; (c) theanti-neoplastic agent and the interferon type II agonist are comprisedwithin a first minicell, and the interferon type I agonist is comprisedwithin a second minicell; (d) the anti-neoplastic agent is comprisedwithin a first minicell, and the interferon type I agonist and theinterferon type II agonist are comprised within a second minicell; or(e) the anti-neoplastic agent is comprised within a first minicell, theinterferon type I agonist is comprised within a second minicell, and theinterferon type II agonist is comprised within a third minicell.

In one embodiment, the composition does not comprise an interferon typeI agonist.

In one embodiment, the anti-neoplastic agent is selected from the groupconsisting of a radionuclide, a chemotherapy drug, a functional nucleicacid, and a polynucleotide from which a functional nucleic acid can betranscribed. In one embodiment, the anti-neoplastic agent is asupertoxic chemotherapy drug. In one embodiment, the supertoxicchemotherapy drug is selected from the group consisting of morpholinylanthracycline, a maytansinoid, ducarmycin, auristatins, calicheamicins(DNA damaging agents), α-amanitin (RNA polymerase II inhibitor),centanamycin, pyrrolobenzodiazepine, streptonigtin, nitrogen mustards,nitrosorueas, alkane sulfonates, pyrimidine analogs, purine analogs,antimetabolites, folate analogs, anthracyclines, taxanes, vincaalkaloids, topoisomerase inhibitors, hormonal agents, and a combinationthereof. In one embodiment, the morpholinyl anthracycline is selectedfrom the group consisting of nemorubicin, PNU-159682, idarubicin,daunorubicin; caminomycin, and oxorubicin. In one embodiment, thesupertoxic chemotherapy drug is PNU-159682.

In one embodiment, the functional nucleic acid is selected from thegroup consisting of a siRNA, a miRNA, a shRNA, a lincRNA, an antisenseRNA, and a ribozyme. In one embodiment, the functional nucleic acidinhibits a gene that promotes tumor cell proliferation, angiogenesis orresistance to chemotherapy and/or that inhibits apoptosis or cell cyclearrest. In some embodiments, the siRNA inhibits ribonucleotide reductaseM1 (RRM1) expression. In some embodiments, the siRNA inhibits Polo likekinase 1 (Plk1) expression. In some embodiments, the miRNA is miRNA16a.

In one embodiment, the interferon type I agonist, the interferon type IIagonist, or the combination of an interferon type I agonist and aninterferon type II agonist is an oligonucleotide. In one embodiment, theoligonucleotide comprises a sequence of at least about 40 nucleotides,at least about 50 nucleotides, or at least about 60 nucleotides. In someembodiments, the oligonucleotide is a polynucleotide product of PNPase1,poly(I:C), poly-ICLC, imiquimod, imidazoquiolineresquimod, cGAMP orCpG-oligodeoxynucleotides.

In one embodiment, the interferon type I agonist is selected from thegroup consisting of double stranded RNA (dsRNA), poly(dA:dT) DNAs,double stranded Z-DNA and B-DNA, DNAs (dsDNAs) longer than 36 bp andDNA-RNA hybrids, bacterial second messenger cyclic-di-GMP, TLR3, TLR4,TLR7, TLR8 and TLR9 agonists, STING agonists, and a combination thereof.

In one embodiment, the interferon type II agonist is selected from thegroup consisting of C-glycosidific form of α-galactosylceramide(α-C-GalCer), α-galactosylceramide (α-GalCer), 12 carbon acyl form ofgalactosylceramide (β-GalCer), β-D-glucopyranosylceramide (β-GlcCer),1,2-Diacyl-3-0-galactosyl-sn-glycerol (BbGL-II), diacylglycerolcontaining glycolipids (Glc-DAG-s2), ganglioside (GD3),gangliotriaosylceramide (Gg3Cer), glycosylphosphatidylinositol (GPI),α-glucuronosylceramide (GSL-1 or GSL-4), isoglobotrihexosylceramide(iGb3), lipophosphoglycan (LPG), lyosphosphatidylcholine (LPC),α-galactosylceramide analog (OCH), threitolceramide, and a combinationthereof. In one embodiment, the interferon type II agonist isα-galactosylceramide (α-GalCer).

In one embodiment, the composition further comprises a bispecific ligandbound to the minicells comprising the anti-neoplastic agent. In oneembodiment, the composition further comprises a bispecific ligand boundto the minicells comprising the type I interferon agonist. In oneembodiment, the composition further comprises a bispecific ligand boundto the minicells comprising the type II interferon agonist.

In one embodiment, the bispecific ligand comprises a first arm thatcarries specificity for a minicell surface structure and a second armthat carries specificity for a non-phagocytotic mammalian cell surfacereceptor. In one embodiment, the minicell surface structure is anO-polysaccharide component of a lipopolysaccharide on the minicellsurface.

In one embodiment, the non-phagocytotic mammalian cell surface receptoris capable of activating receptor-mediated endocytosis of the minicell.

In one embodiment, the bispecific ligand comprises a bispecific antibodyor antibody fragment. In one embodiment, the antibody or antibodyfragment comprises a first multivalent arm that carries specificity fora bacterially derived minicell surface structure and a secondmultivalent arm that carries specificity for a cancer cell surfacereceptor, wherein the cancer cell surface receptor is capable ofactivating receptor-mediated endocytosis of the minicell.

In one embodiment, the composition comprises fewer than about 1contaminating parent bacterial cell per 10⁷ minicells, fewer than about1 contaminating parent bacterial cell per 10 minicells, fewer than about1 contaminating parent bacterial cell per 10⁹ minicells, fewer thanabout 1 contaminating parent bacterial cell per 10¹⁰ minicells, or fewerthan about 1 contaminating parent bacterial cell per 10¹¹ minicells.

In one embodiment, the composition further comprises a pharmaceuticallyacceptable carrier. In one embodiment, the minicells are approximately400 nm in diameter. In one embodiment, the composition is free of parentbacterial cell contamination removable through 200 nm filtration.

In one embodiment, the composition comprises the following amount ofminicells or killed bacterial cells (a) at least about 10⁹; (b) at leastabout 1×10⁹; (c) at least about 2×10⁹; (d) at least about 5×10⁹; (e) atleast 8×10⁹; (f) no more than about 10¹¹; (g) no more than about 1×10¹¹;(h) no more than about 9×10¹⁰; or (i) no more than about 8×10¹⁰.

One embodiment of the invention relates to a method of treating asubject in need, comprising administering to the subject an effectiveamount of a composition disclosed herein. In one embodiment, the subjectis a human, a non-human primate, a dog, a cat, a cow, a sheep, a horse,a rabbit, a mouse, or a rat. In one embodiment, the subject is a human.

In one embodiment, the subject is suffering from a cancer. In oneembodiment, the cancer is selected from the group consisting of lungcancer, breast cancer, brain cancer, liver cancer, colon cancer,pancreatic cancer, and bladder cancer. In one embodiment, the cancer isselected from the group consisting of an acute lymphoblastic leukemia;acute myeloid leukemia; adrenocortical carcinoma; AIDS-related cancers;AIDS-related lymphoma; anal cancer; appendix cancer; astrocytomas;atypical teratoid/rhabdoid tumor; basal cell carcinoma; bladder cancer;brain stem glioma; brain tumor; breast cancer; bronchial tumors; Burkittlymphoma; cancer of unknown primary site; carcinoid tumor; carcinoma ofunknown primary site; central nervous system atypical teratoid/rhabdoidtumor; central nervous system embryonal tumors; cervical cancer;childhood cancers; chordoma; chronic lymphocytic leukemia; chronicmyelogenous leukemia; chronic myeloproliferative disorders; coloncancer; colorectal cancer; craniopharyngioma; cutaneous T-cell lymphoma;endocrine pancreas islet cell tumors; endometrial cancer;ependymoblastoma; ependymoma; esophageal cancer; esthesioneuroblastoma;Ewing sarcoma; extracranial germ cell tumor; extragonadal germ celltumor; extrahepatic bile duct cancer; gallbladder cancer; gastric(stomach) cancer; gastrointestinal carcinoid tumor; gastrointestinalstromal cell tumor; gastrointestinal stromal tumor (GIST); gestationaltrophoblastic tumor; glioma; hairy cell leukemia; head and neck cancer;heart cancer; Hodgkin lymphoma; hypopharyngeal cancer; intraocularmelanoma; islet cell tumors; Kaposi sarcoma; kidney cancer; Langerhanscell histiocytosis; laryngeal cancer; lip cancer; liver cancer;malignant fibrous histiocytoma bone cancer; medulloblastoma;medulloepithelioma; melanoma; Merkel cell carcinoma; Merkel cell skincarcinoma; mesothelioma; metastatic squamous neck cancer with occultprimary; mouth cancer; multiple endocrine neoplasia syndromes; multiplemyeloma; multiple myeloma/plasma cell neoplasm; mycosis fungoides;myelodysplastic syndromes; myeloproliferative neoplasms; nasal cavitycancer; nasopharyngeal cancer; neuroblastoma; Non-Hodgkin lymphoma;nonmelanoma skin cancer; non-small cell lung cancer; oral cancer; oralcavity cancer; oropharyngeal cancer; osteosarcoma; other brain andspinal cord tumors; ovarian cancer; ovarian epithelial cancer; ovariangerm cell tumor; ovarian low malignant potential tumor; pancreaticcancer; papillomatosis; paranasal sinus cancer; parathyroid cancer;pelvic cancer; penile cancer; pharyngeal cancer; pineal parenchymaltumors of intermediate differentiation; pineoblastoma; pituitary tumor;plasma cell neoplasm/multiple myeloma; pleuropulmonaryblastoma; primarycentral nervous system (CNS) lymphoma; primary hepatocellular livercancer; prostate cancer; rectal cancer; renal cancer; renal cell(kidney) cancer; renal cell cancer; respiratory tract cancer;retinoblastoma; rhabdomyosarcoma; salivary gland cancer; Sezarysyndrome; small cell lung cancer; small intestine cancer; soft tissuesarcoma; squamous cell carcinoma; squamous neck cancer; stomach(gastric) cancer; supratentorial primitive neuroectodermal tumors;T-cell lymphoma; testicular cancer; throat cancer; thymiccarcinoma;thymoma; thyroid cancer; transitional cell cancer; transitional cellcancer of the renal pelvis and ureter; trophoblastic tumor; uretercancer; urethral cancer; uterine cancer; uterine sarcoma; vaginalcancer; vulvar cancer; Waldenstrom macroglobulinemia; and Wilm's tumor.

In one embodiment, the brain cancer or tumor is selected from the groupconsisting of brain stem glioma, central nervous system atypicalteratoid/rhabdoid tumor, central nervous system embryonal tumors,astrocytomas, craniopharyngioma, ependymoblastoma, ependymoma,medulloblastoma, medulloepithelioma, pineal parenchymal tumors ofintermediate differentiation, supratentorial primitive neuroectodermaltumors and pineoblastoma.

In one embodiment, the composition is administered at least once a weekover the course of several weeks. In one embodiment, the composition isadministered at least once a week over several weeks to several months.In one embodiment, the composition is administered at least once a weekfor about 2, about 3, about 4, about 5, about 6, about 7, about 8, about9, about 10, about 11, about 12, about 13, about 14, about 15, about 16,about 17, about 18, about 19 or about 20 weeks or more. In oneembodiment, the composition is administered about twice every week. Inone embodiment, the composition is administered twice a week for about2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about10, about 11, about 12, about 13, about 14, about 15, about 16, about17, about 18, about 19 or about 20 weeks or more.

Both the foregoing summary and the following description of the drawingsand detailed description are exemplary and explanatory. They areintended to provide further details of the invention, but are not to beconstrued as limiting. Other objects, advantages, and novel featureswill be readily apparent to those skilled in the art from the followingdetailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical depiction of an EnGeneIC Dream Vehicle (EDV)(e.g., a bacterial minicell) comprising a bispecific antibody forO-polysaccharide and human epidermal growth factor receptor antigens andloaded with the anti-cancer drug PNU-159682 (an anthracycline analogue).

FIG. 2 is a graphical depiction of an EnGeneIC Dream Vehicle (EDV)comprising O-polysaccharides on the surface and loaded withimmunomodulatory 60mer double stranded DNA.

FIG. 3 is a graphical depiction of an EnGeneIC Dream Vehicle (EDV)comprising O-polysaccharides on the surface and loaded withimmunomodulatory alpha galactosylceramide (ctGC).

FIG. 4 is a graphical summary of a clinical trial evaluatingEGFR-targeted and miRNA16a loaded EDVs for treating mesotheliomapatients.

FIGS. 5A-5B shows the cytotoxic effect of the indicated chemotherapydrugs on an A549 lung cancer cell line. FIG. 5[ ]A compares the effectof the indicated chemotherapy drugs with the supertoxic drug PNU-159682.FIG. 5[ ]B compares the effect of doxorubicin and PNU-159682.

FIGS. 6A-6B shows the effect of the indicated chemotherapy drugs onadreno-cortical cancer cell lines ACC01 (FIG. 6A) and ACC07 (FIG. 6B).

FIG. 7 shows the effect of the indicated chemotherapy drugs on theMDA-MB-468 breast cancer cell line.

FIGS. 8A-8B shows the effect of the indicated chemotherapy drugs on thehuman colorectal cancer cell lines Caco-2 (FIG. 8A) and HCT116 (FIG.8B).

FIG. 9 shows the effect of the indicated chemotherapy drugs on theglioblastoma cell line U87-MG.

FIGS. 10A-10B shows the effect of the indicated chemotherapy drugs onthe human pancreatic cell lines MiaPaca-2 (FIG. 10A) andgemicitabine-resistant MiaPaca-2 GemR cells (FIG. 10B).

FIG. 11 shows the effect of EDVs targeted to EGFR and loaded withPNU-159682 (^(EGFR)EDVs₆₈₂™) or doxorubicine (^(EGFR)EDVs_(Dox)™) onA549 xenograft tumor growth in mice. Negative controls are saline onlyor untargeted EDVs loaded with PNU-159682 (EDVs₆₈₂™). Arrows indicatedwhen the mice were treated with the indicated saline or EDVcompositions. Asterisks indicate when the mice initially treated withsaline were administered the ^(EGFR)EDVs₆₈₂™ composition.

FIG. 12 shows expression of GAPDH (glyceraldehyde 3-phosphatedehydrogenase) (G), KSP (Kinesin Spindle Protein), Plk1 (Polo likekinase 1) (P), and RRM1 (ribonucleotide reductase enzyme 1) (R) relativeto GAPDH expression in the indicated NSCLC cell lines.

FIGS. 13A-13B shows the effect of delivering EGFR-targeted,siRRM1-packaged EDVs to a mesothelioma cell line (MSTO, FIG. 13A) or anadreno-cortical cancer cell line (H295R, FIG. 13B).

FIG. 14 shows the effect of delivering EGFR-targeted, miRNA16a(^(EGFR)EDV_(miRNA16a)™), or EGFR-targeted, siRRM1-packaged EDVs(^(EGFR)EDV_(siRRM1)™), on mesothelioma xenograft tumor growth in Balb/cnu/nu mice. Negative controls were saline or EGFR-targeted EDVs loadedwith scrambled siRNA.

FIG. 15 shows tumors isolated from mesothelioma xenograft Balb/c nu/numice treated with EGFR-targeted, miRNA16a (^(EGFR)EDV_(miRNA16a)™), orEGFR-targeted, siRRM1-packaged EDVs (^(EGFR)EDV_(siRRM1)™), as comparedto saline treated or EGFR-targeted EDVs loaded with scrambled siRNA.

FIGS. 16A and 16B show apoptosis induced in adreno-cortical cancer cells(ACC01) by EGFR-targeted EDVs loaded with siRNA targeting Polo likekinase 1 (^(EGFR)EDV™_(siPLK)™) and ribonucleotide reductase enzyme 1(^(EGFR)EDV_(siRRM1)™) based on measuring the number of cellular debris(FIG. 16A) and the ratio of Annexin5 to Propidium Iodide (PI) positivecells (FIG. 16B). Apoptosis in untreated ACC01 cells, in ACC01 cellstreated with EDVs loaded with irrelevant siRNA(^(EGFR)EDV™_(siLuciferase)™), and unloaded EDVs (^(EGFR)EDV™) areincluded as negative controls.

FIGS. 17A-D show sub-G1 arrest (FIG. 17D) induced in adreno-corticalcancer cells (ACC01) by EGFR-targeted EDVs loaded with siRNA targetingPolo like kinase 1 (^(EGFR)EDV™_(siPLK)™) (FIG. 17D) and ribonucleotidereductase enzyme 1 (^(EGFR)EDV_(siRRM1)™) based on measuring the numberof cellular debris and the ratio of Annexin5 to Propidium Iodide (PI)positive cells. Apoptosis in untreated ACC01 cells (FIG. 17A), in ACC01cells treated with EDVs loaded with irrelevant siRNA(^(EGFR)EDV™_(siLuciferase)™) (FIG. 17C), and unloaded EDVs(^(EGFR)EDV™) (FIG. 17B) are included as negative controls.

FIG. 18 shows the effect of A549 (lung cancer) xenograft tumor growth inBalb/c nu/nu mice treated with: (i) solidtriangle=^(EGFR)EDVs_(PNU-159682)™+EDVs_(40mer)™, (ii) solidcircle=^(EGFR)EDVs_(PNU-59682)™, (iii) opensquare=^(EGFR)EDVs_(PNU-159682)™+EDVs, (iv) opentriangle=^(EGFR)EDVs_(PNU-159682)™+EDVs_(50mer)™, and (v) solidsquare=saline. The mice were treated with these EDVs combinations at day24, 27, 29, 31, 34, 36, and 38 after the xenograft implantation asindicated with up arrows. On days 36 and 38, the saline group mice withtumor volume of ˜650 mm³ were treated with^(EGFR)EDVs_(PNU-159682)™+EDVs_(50mer)™ as indicated by the down arrows.

FIG. 19 shows the effect on A549 (lung cancer) xenograft tumor growth inBalb/c nu/nu mice treated with EDVs comprising 40mers(^(EGFR)EDVs_(40mers)™) in combination with EDVs comprising PNU-159682(^(EGFR)EDVs_(PNU)™). The triangles indicate treatment days.

FIG. 20 shows the effect on A549 (lung cancer) xenograft tumor growth inBalb/c nu/nu mice treated with saline (negative control), IFN-γ (0.5×10⁴IU per dose), EGFR-targeted EDVs loaded with doxorubicine(^(EGFR)EDVs_(Dox)™), and ^(EGFR)EDVs_(Dox)™+IFN-γ. The trianglesindicate treatment days.

FIG. 21 shows the effect on MDA-MB 468 (breast cancer) xenograft tumorgrowth in Balb/c nu/nu mice treated with saline (negative control),IFN-γ (0.5×104 IU per dose), EGFR-targeted EDVs loaded with doxorubicine(^(EGFR)EDVs_(Dox)™), and ^(EGFR)EDVs_(Dox)™+IFN-γ. The trianglesindicate treatment days.

FIG. 22 shows another experiment illustrating the effect on MDA-MB 468(breast cancer) xenograft tumor growth in Balb/c nu/nu mice treated withsaline (negative control), IFN-γ (0.5×104 IU per dose), EGFR-targetedEDVs loaded with doxorubicine (^(EGFR)EDVs_(Dox)™), and^(EGFR)EDVs_(Dox)™+IFN-γ. The triangles indicate treatment days.

FIG. 23 shows the effect on doxorubicin-resistant A549 xenograft tumorgrowth in Balb/c nu/nu mice treated with saline (negative control, Group1), EGFR-targeted EDVs loaded with doxorubicine (^(EGFR)EDVs_(Dox)™,Group 2), ^(EGFR)EDVs_(Dox)™+IFN-γ (0.75×10⁴ IU per dose) (Group 3), and^(EGFR)EDVs_(Dox)™+IFN-γ (0.5×10⁴ IU per dose) (Group 4). Mice in Groups1-3 received treatment twice per week indicated by the solid triangles.Mice in Group 4 were treated three times per week as indicated by opentriangles.

FIGS. 24A-K show a cytokine profile of patients from a First-in-Manclinical study where different dosages of ^(EGFR)EDVs™ loaded withpaclitaxel were administered. (FIG. 24A)=pg/mL of IL-6 measured for eachof the 5 doses; (FIG. 24B)=pg/mL of IL-8 measured for each of the 5doses; (FIG. 24C)=pg/mL of IL-10 measured for each of the 5 doses; (FIG.24D)=pg/mL of TNF-α measured for each of the 5 doses; (FIG. 24E)=pg/mLof IFN-α measured for each of the 5 doses; (FIG. 24F)=pg/mL of IFN-γmeasured for each of the 5 doses; (FIG. 24G)=pg/mL of IL-113 measuredfor each of the 5 doses; (FIG. 24H)=pg/mL of IL-2 measured for each ofthe 5 doses; (FIG. 24I)=pg/mL of IL-4 measured for each of the 5 doses;and (FIG. 24J)=pg/mL of IL-12 measured for each of the 5 doses. Finally,(FIG. 24K) shows the five doses tested.

FIGS. 25A-K show a cytokine profile of patients from a First-in-Manclinical study where different dosages of ^(EGFR)EDVs™ loaded withdoxorubicin were administered. (FIG. 25A)=pg/mL of IL-6 measured foreach of the 8 doses; (FIG. 25B)=pg/mL of IL-8 measured for each of the 8doses; (FIG. 25C)=pg/mL of IL-10 measured for each of the 8 doses; (FIG.25D)=pg/mL of TNF-α measured for each of the 8 doses; (FIG. 25E)=pg/mLof IFN-α measured for each of the 8 doses; (FIG. 25F)=pg/mL of IFN-γmeasured for each of the 8 doses; (FIG. 25G)=pg/mL of IL-113 measuredfor each of the 8 doses; (FIG. 25H)=pg/mL of IL-2 measured for each ofthe 8 doses; (FIG. 25I)=pg/mL of IL-4 measured for each of the 8 doses;and (FIG. 25J)=pg/mL of IL-12 measured for each of the 8 doses. Finally,(FIG. 25K) shows the additional three doses tested, with the first fivedoses being the same as those shown in (FIG. 24K).

FIG. 26 shows signaling pathways of cytosolic DNA sensors with DNAchallenge. Up to now, many cytosolic DNA sensors have been defined todetect intracellular double-stranded DNAs. RNA polymerase IIItranscribes AT-rich DNAs into RNAs that are recognized by RNA sensorRIG-I, followed by STING and IRF3 activation. DNA sensors DAI, IFI16,DDX41 and LSm14A sense dsDNA directly to activate STING for type I IFNproduction. In the presence of dsDNAs, cGAS catalyzes the synthesis ofcGAMP, a strong activator of STING. With dsDNAs, LRRFIP1 initiatesβ-catenin and IRF3 activation in a STING-dependent manner. Other DNAsensors prime immune responses independently of STING. After recognitionof dsDNAs, Sox2 triggers the activation of the Tab2/TAK1 complex inneutrophils. When detected by dsDNAs, DHX9/36 activates NFκB and IRF7through MyD88. DNA sensor Ku70 triggers the activation of IRF1 and IRF7.AIM2 initiates the activation of inflammasome through ASC with DNAbinding.

FIGS. 27A-27L shows RAW264.7 cell and bone marrow derived dendritic cell(BMDC) activation in response to EDV treatment. (FIG. 27 A) CD86expression in RAW cells incubated directly with 1 μg/ml LPS, Ep-EDV,Ep-EDV682, or 682. (FIG. 27 B) CD86 expression in RAW cells co-culturedwith 4T1 or CT26Ep12.1 cells treated with Ep-EDV, Ep-EDV682, or 682. RAWcells co-cultured with Ep-EDV682 treated tumor cells resulted in asignificant increase in CD86 expression. (FIG. 27 C) TNFα production inRAW cell/tumor cell co-cultures showing a significant increase in TNFαproduction by RAW cells incubated with EDV treated tumor cells. (FIG. 27D) IL-6 production in RAW cell/tumor cell co-cultures showing asignificant increase in IL-6 production by RAW cells incubated with EDVtreated tumor cells. (FIG. 27 E) PCR quantitation of IFNα and IFNβexpression in BMDC/4T1 co-cultures. (FIG. 27 F) PCR quantitation of IFNαand IFNβ expression in BMDC/CT26Ep12.1 co-cultures. Quantitation of(FIG. 27 G) CD86^(Hi) and MHC Class II^(Hi) expression and (FIG. 27 H)CD80^(H)i expression in BMDC/tumor cell co-cultures. (FIG. 27 I) Flowcytometic density plots of MHC Class II vs CD86 expression in BMDCco-cultures with EDV and drug-treated CT26Ep12.1 cells. ELISA analysisof (FIG. 27 J) TNFα (FIG. 27 K) IL-12p40 and (FIG. 27 L) IL-6 from thesupernatants of BMDC/tumor cell co-cultures. Data represents mean±s.e.m.and analyzed by one-way ANOVA and Tukey's multiple comparison test.

FIGS. 28A-28H shows tumor response and macrophage activation in responseto EDV treatment. Tumor growth in response to Ep-EDV and Ep-EDV682treatment in BALB/c mice bearing (FIG. 28 A) 4T1 or (FIG. 28 B)CT26Ep12.1 tumors. Tumor growth in response to (FIG. 28 C) EDV-682 andEDV-EGFR682 in BALB/C nude mice T84 xenografts and (FIG. 28 D) EDV-682,EDV-EGFRDox, and EDV-EGFR682 in BALB/C nude mice bearing A549/MDRxenografts. Green arrow indicates where EDV-EGFR682 treatment offormally saline treated mice was begun. Data (FIGS. 28A-D) representsmean±s.e.m. and analyzed by a two way ANOVA and Tukey's multiplecomparison test (FIG. 28E) xCELLigence RTCA of CD11b⁺ isolated from 4T1tumors and co-cultured with 4T1 cells at a 5:1 (E:T) ratio. Plotrepresents normalized cell index which correlates to cell adhesion andgrowth/death vs time. CD11b⁺ cells from 4T1 tumors undergo and initialadhesion and settling phase as indicated by an increase in cell index,followed by growth or death represented by an increasing or decreasingcell index. (FIG. 28 F) Ratio of M1 (CD86+): M2 (CD206⁺) macrophages in4T1 tumors of treated mice. (FIG. 28 G) xCELLigence RTCA of CD11b⁺isolated from CT26Ep12.1 tumors and co-cultured with CT26Ep12.1 cells ata 5:1 (E:T) ratio. (FIG. 28H) Ratio of M1 (CD86+): M2 (CD206⁺)macrophages in CT26Ep12.1 tumors of treated mice. Data (FIGS. 28F and28H) represents mean±s.e.m. and analyzed by one way ANOVA and Tukey'smultiple comparison test.

FIGS. 29A-29J shows NK cell response to EDV treatment. (FIG. 29 A)xCELLigence RTCA of NK cells isolated from spleens of mice bearing 4T1tumors co-cultured with 4T1 cells at a 20:1 (E:T) ratio. Plot representscell viability, calculated from the normalized cell index, over time.(FIG. 29B) % viability of 4T1 cells co-cultured with NK cells fromsaline, Ep-EDV, or Ep-EDV-682 treated mice 70 h following the additionof NK. (FIG. 29C) xCELLigence RTCA of NK cells isolated from spleens ofmice bearing CT26Ep12.1 tumors co-cultured with CT26Ep12.1 cells at a20:1 (E:T) ratio. (FIG. 29D) % viability of CT26Ep12.1 cells co-culturedwith NK cells from saline, Ep-EDV, or Ep-EDV-682 treated mice 50 hfollowing the addition of NK cells (FIG. 29E) Expression of NKG2D in NKcells (CD45+, CD11b+, DX5+) within 4T1 tumors showing an increase inNKG2D expression in Ep-EDV-682 treated mice. Production of (FIG. 29F)RANTES and (FIG. 29G) TNFα in co-cultures of NK cells isolated from thespleens of EDV treated mice bearing 4T1 tumors with 4T1 cells. (FIG.29H) Quantitation of NKG2D ligands RAE-1, H60a, and MULT-1 on thesurface of 4 different mouse tumor cell lines. (FIG. 29I) xCELLigenceRTCA of NK cells isolated from spleens of EpEDV-682 treated mice bearing4T1 tumors co-cultured with 4T1 cells at a 20:1 (E:T) ratio in thepresence of RAE-1 and/or H60a inhibiting antibodies demonstrating thatboth are important in NK tumor cell cytolysis. (FIG. 29J) Quantitationof NK cytolysis 80 h post NK cell addition showing significantinhibition of NK cytolysis with H60a antibody alone of in conjunctionwith RAE-1 inhibiting antibodies. Data (FIGS. 29B, 29D, 29E-G, and 29J)represents mean±s.e.m. and analyzed by one way ANOVA and Tukey'smultiple comparison test.

FIGS. 30A-30G shows interstitial tumor cytokine/chemokine production andcytokine production by splenocyte/tumor cell co-cultures in response toEDV treatment. ELISA analysis of interstitial cytokines and chemokinesproduced in response to Ep-EDV and Ep-EDV682 treatment in BALB/c micebearing a (FIG. 30A) 4T1 or (FIG. 30B) CT26Ep12.1 tumors. Ep-EDV-682treatment results in an increase in predominantly Th1 cytokines. Datarepresents mean±s.e.m. Individual cytokine data analyzed by one wayANOVA and Tukey's multiple comparison test. ELISA analysis of (FIG. 30C)TNFα (FIG. 30D) IL-2 (FIG. 30E) IL-1β (FIG. 30F) IFNγ and (FIG. 30G)IL-10 from the supernatants of co-cultures of splenocytes isolated fromsaline, Ep-EDV, and Ep-EDV-682 treated mice bearing 4T1 and CT26Ep12.1tumors with their corresponding tumor cells. Data represents mean±s.e.m.One way ANOVA analysis and Tukey's multiple comparison used to comparegroups + or − tumor cells. T-test used to compare individual treatmentswith and without tumor cells.

FIGS. 31A-31I shows T-cell function and phenotype in response to EDVtreatment. (FIG. 31A) xCELLigence RTCA of CD8⁺ T-cells isolated from thespleens of mice bearing 4T1 tumors co-cultured with 4T1 cells at a 30:1(E:T) ratio. Plot represents normalized cell index which correlates tocell adhesion and growth/death vs time. (FIG. 31B) % viability of 4T1cells co-cultured with CD8⁺ T-cells from saline, Ep-EDV, or Ep-EDV-682treated mice 30 h following the addition of CD8⁺ T-cells. (FIG. 31C)xCELLigence RTCA of CD8⁺ T-cells isolated from the spleens of micebearing CT26Ep12.1 tumors co-cultured with CT26Ep12.1 cells at a 30:1(E:T) ratio. Plot represents normalized cell index which correlates tocell adhesion and growth/death vs time. (FIG. 31D) % viability ofCT26Ep12.1 cells co-cultured with CD8⁺ T-cells from saline, Ep-EDV, orEp-EDV-682 treated mice 20 h following the addition of CD8⁺ T-cells.(FIG. 31E) Percentage of CD8⁺ T-cells (defined as CD45⁺, CD3⁺, CD8⁺)detected in 4T1 tumors. (FIG. 31F) Percentage of T-regs (defined asCD45⁺, CD3⁺, CD4⁺, CD25⁺) detected in 4T1 tumors. (FIG. 31G) Numbers ofT-cells in tumor draining lymph nodes of mice bearing 4T1 tumors shownas % of total cells. (FIG. 31H) % CD80/MHC Class II expression indendritic cells in the tumor draining lymph nodes of mice bearing 4T1tumors. (FIG. 31I) Confocal image of the interaction between isolatedCD8⁺ T-cells from Ep-EDV-682 treated mice with 4T1 cells. Red—actin,green—perforin, blue (dark)—DAPI; scale bar 10 μm. Data (FIGS. 31B, 31D,and 31E-H) represents mean±s.e.m. and analyzed by one way ANOVA andTukey's multiple comparison test (FIGS. 31B, 31D, and 31E-G) or t-test(FIG. 31H).

FIGS. 32A-32G shows Prognostic Indicators and Immunophenotyping ofpatient peripheral blood mononuclear cells (PBMCs) reveals evidence ofenhanced antigen presentation by dendritic cells and monocytes andelevated cytotoxic CD8+ T cell content at dose 12. Prognostic indicators(FIG. 32A) CA19-9 and (FIG. 32B) C-reactive protein serum levels.Analysis of PBMC with Duraclone immunophenotyping panels for (FIG. 32C)Monocytes and (FIG. 32D) intermediate (CD14+CD16++) antigen presentingmonocyte subtype. Expressed as % Leukocytes. Dendritic cell subtypesincluding (FIG. 32E) myeloid dendritic (Clec9A+) cells (mDC) that drivethe CD8+ Effector T cell response and (FIG. 32F) Plasmacytoid dendriticand myeloid dendritic (professional antigen presenting DC). Expressed as% DC or % mDC as indicated. (FIG. 32G) CD8+ T cell subtypes. CytotoxicCD8+ T cells include effectors and exhausted (PD1+) subtypes.

FIGS. 33A-33I shows a schematic of how the EDV first creates animmunogenic tumor microenvironment via the delivery of cytotoxic agentsdirectly to the tumor, then stimulates the innate immune system eitherdirectly or indirectly towards an antitumor phenotype, and finallyproduces an adaptive response in which tumor specific cytotoxic T-cellsarise. (FIG. 33A) Ep-EDV-682 enters the tumor microenvironment via theleaky vasculature resulting in tumor cell apoptosis and the release ofimmune activating DAMPs. (FIG. 33B) The interaction of macrophageswithin the tumor microenvironment via engulfment of apoptotic cells oreven EDVs directly, results M1 macrophage polarization and release ofinflammatory cytokines TNFα and IL-6 (FIG. 33C) M1 macrophages arecapable of further lysing tumor cells and release MIP-1α which canrecruit additional immune cells. (FIG. 33D) Immature dendritic cellsengulf apoptotic cell bodies and released tumor antigens which occur inresponse to Ep-EDV-682 treatment and mature releasing type 1interferons, TNFα, IL-12p40 and IL-6. (FIG. 33E) Mature DC then migrateto the lymph node for antigen presentation to T-cells. (FIG. 33F) NKcell activation also occurs in the tumor microenvironment resulting inrelease of IFNγ and TNFα as well as RANTES to attract additional immunecells. Further, activated NK cells effectively lyse tumor cells. (FIG.33G) The release of RANTES and MIP-1α recruits additional T-cells, NKcells, and macrophages into the tumor where (FIG. 33H) tumor specificCD8⁺ T-cells then contribute the response via tumor cell lysis. (FIG.33I) All of these steps combine to create and effective antitumor immuneresponse.

FIGS. 34A-34E shows RAW264.7 and JAWS II cell activation in response toEDV treatment. (FIG. 34A) TNFα production by RAW264.7 cells incubateddirectly with EDV formulations. (FIG. 34B) IL-6 productions by RAW264.7cells incubated directly with EDV formulations. (FIG. 34 C) Flowcytometric histogram overlays of CD86 and MHC Class II expression inJAWS II cells co-cultures with untreated CT26Ep12.1 and 4T1 cells orthose treated with Ep-EDV, Ep-EDV-682, or 682 alone. (FIG. 34D)Quantitation of CD86 expression as determined via flow cytometry on JAWSII cells co-cultured with treated tumor cells. (FIG. 34E) Quantitationof MHC Class II expression as determined via flow cytometry on JAWS IIcells co-cultured with treated tumor cells. Data represent mean±s.e.m.and are analyzed by one-way ANOVA and Tukey's multiple comparison test.

FIGS. 35A-35I shows body weight changes and macrophage activation inresponse to EDV treatment in Balb/c and Balb/c nude xenografts. % changein body weights of mice bearing (FIG. 35A) CT26Ep12.1 (FIG. 35 B) 4T1(FIG. 35 C) T84 and (FIG. 35 D) A549/MDR tumors in response totreatment. No more than 5% weight loss is seen with the initial dose,and weights then recover and stabilize with subsequent dosing. Datarepresents mean±s.e.m. M1/M2 (CD86:CD206) ratio of macrophages in (FIG.35E) A549/MDR and (FIG. 35F) T84 tumors of EDV treated mice. (FIG. 35 G)xCELLigence RTCA of CD11b⁺ isolated from A549/MDR tumors and co-culturedwith A549/MDR cells at a 5:1 (E:T) ratio. Plot represents normalizedcell index which correlates to cell adhesion and growth/death vs time.(FIG. 35H) % cytolysis of A549/MDR cells co-cultured with CD11b⁺ cellsfrom the tumors of saline or EGFR-EDV-682 treated mice 6.5 h followingthe addition of CD11b⁺ cells. (FIG. 35I) Production of MIP-1α inco-cultures of CD11b⁺ cells isolated from treated 4T1 tumors with 4T1cells. Data (FIGS. 35E, 35F, 35H, and 35I) represents mean±s.e.m. andanalyzed by one way ANOVA and Tukey's multiple comparison test (FIG.35E, 35H, 35I) or t-test (FIG. 35F).

FIGS. 36A-36C shows NK cell response to EDV treatment. (FIG. 36A)xCELLigence RTCA of NK cells isolated from spleens of Balb/c nude micebearing T84 tumors co-cultured with T84 cells at a 10:1 (E:T) ratio.Plot represents cell viability, calculated from the normalized cellindex, over time. (FIG. 36B) Granzyme B production in co-cultures of NKcells isolated from spleens of saline and EGFR-EDV-682 treated mice andT84 cells. Data represents mean±s.e.m. and analyzed by t-test. (FIG.36C) xCELLigence RTCA of NK cells isolated from spleens of Balb/c nudemice bearing A549/MDR tumors co-cultured with A549/MDR cells at a 10:1(E:T) ratio. Plot represents cell viability, calculated from thenormalized cell index, over time (Saline n=5; EGFR-EDV-682 n=4).

FIGS. 37A-37C shows receptor expression and drug sensitivity screeningof patient derived pancreatic ductal adenocarcinoma cells. (FIG. 37A)EGFR surface receptor quantitation of cells from the head of the tumor.(FIG. 37B) EGFR surface receptor quantitation of cells from the tail ofthe tumor. (FIG. 37C) Drug sensitivity and IC50 of first and second linechemotherapy drugs/drug combinations as compared to 682 sensitivity.

FIG. 38 shows single cells from the total PBMC pool (FSC v SSC) weregated based on forward scatter width (FSC-W) versus forward scatter area(FSC-A) then analysed for CD45 staining (CD45+ gate on FSC v CS45+).Cell viability was 96% (Count and viability kit #C00162, BeckmanCoulter, data not shown) with dead cells excluded based on a FSCthreshold discriminator of 80. Total dendritic cells (DCs) were gated onLeukocytes (CD45+) and defined as HLA-DR+ and Lineage− (#B53351, BeckmanCoulter). The lineage negative marker was comprised of a pool ofantibodies conjugated with the same fluorophore (PE) raised against CD3,CD14, CD19, CD20 and CD56 used to negatively select for T cells,Monocytes, B cells, and NK cells, respectively. The remaining cells thatwere HLA-DR+ were gated as dendritic and subdivided into plasmacytoidDCs (CD11c-CD123+) or antigen presenting myeloid DCs (CD11c+CD123−). Themyeloid DCs (mDC) were divided into the three major subsets, CD1c+ mDC1,CD141+ mDC2 (Clec9A+ shown here) and CD16+ mDCs. CD14+ expressiondefined the monocytes (#B93604, Beckman Coulter) that were gated onleukocytes, then subdivided into classical (CD14+CD16-), intermediate(CD14+CD16+) and non-classical (CD14+CD16++). T cells (#B53328, BeckmanCoulter) were CD3+ and gated on lymphocytes (CD45+ SSC low). CD3+ T cellsubsets, T helper (CD4+) and cytotoxic T (CD8+) were then defined. ThePD1+CD8+ cytotoxic T cell gate was plotted against SSC and gated on CD8+T cells. All FSC and SSC axes are linear while fluorescence channel axes(all CD markers) are logarithmic or bi-exponential (‘logicle’, Kaluzasoftware, Beckman Coulter).

FIGS. 39A-39C shows Flow Cytometric analysis showing purity of cellisolations used for xCelligence RTCA experiments. (FIG. 39A) Isolationof CD11b⁺ cells from mouse tumors. Density plots showing isotype controlvs. FSC and CD11b vs. FSC. Samples had ˜80% purity of CD11b⁺ cells.(FIG. 39B) Isolation of NK cells from mouse spleens. Density plots showisotype control vs. FSC, NKp46 vs. FSC and CD11b vs. FSC. Samples had˜90% purity of NK cells. (FIG. 39C) CD8⁺ T-cell isolation from mousespleens. Density plots show isotype control vs. FSC, CD3e vs. FSC, andCD8 vs. FSC. Samples had ˜90% purity of T-cells (CD3e⁺) with over 98% ofthose T-cells being CD8⁺.

FIG. 40 shows combination treatment using ^(Ep)minicell_(Dox) andminicell_(α-GC) in a syngeneic mouse model (^(Ep)CT26 colon tumors inBalb/c mice).

FIG. 41 shows combination treatment of ^(Ep)minicell_(D)ox andminicell_(α-GC) is effective in reducing large tumors in Balb/c micebearing CT26 isograft.

FIG. 42 shows effect of ^(Ep)minicell_(Dox) and minicell_(α-GC) on tumorregression in Balb/c mice with CT26 isograft.

FIGS. 43A-43F shows different sized CT26 isografts treated with (FIGS.43A and 43B)^(EP)minicell_(Dox) and minicell_(α-GC), (FIGS. 43D and 43E)minicell_(α-GC) only, (FIG. 43F) ^(Ep)minicell_(Dox) only, and (FIG.43C) saline.

FIGS. 44A-44B shows different sized CT26 isografts treated with^(EP)minicell_(Dox) and minicell_(α-GC).

FIGS. 45A-45E shows aGC-CD1D presentation of JAWSII Cells Followingminicell_(α-GC) treatment at various time points (FIGS. 45A-E)

DETAILED DESCRIPTION I. Overview

The present invention is based on the discovery that compositionscomprising a combination of (i) an anti-neoplastic agent and a type Iinterferon agonist; (ii) an anti-neoplastic agent and a type IIinterferon agonist; or (iii) an anti-neoplastic agent, a type Iinterferon agonis, and a type II interferon agonist, wherein at leastthe anti-neoplastic agent is packaged within intact bacterially derivedminicells, can synergistically improve cancer treatment strategies.

The combination of active agent and immunomodulatory agent(s), where atleast the anti-neoplastic agent, and optionally the type I and/or typeII interferon agonist is packaged within intact bacterially derivedminicells, results in dramatic efficacy against cancerous cells, as wellas surprising lack of drug-resistance development in subjects. Thedescribed compositions avoid the toxicity associated with systemicdelivery of anti-neoplastic drugs combined with immunomodulatory drugssuch as type I and/or type II interferon agonists to providesynergistically improved cancer treatment strategies.

Recent advances in cancer immunotherapy have resulted in unprecedented,durable clinical responses in specific cancers (Emens et al., 2017;Farkona et al., 2016; Oiseth and Aziz, 2017; Sharma et al., 2017;Ventola, 2017). However, current immunotherapeutic strategies haveresulted in limited success rates across a variety of tumor types and asignificant proportion of patients who initially demonstrate encouragingtumor regression relapse over time (Emens et al., 2017; Mellman et al.,2011; Oiseth and Aziz, 2017; Sharma et al., 2017; Ventola, 2017).

Described in Example 16 below is data elucidating the mechanism of thecyto-immuno-therapy function of a tumor-targeted nanocell therapeutic,where it launches a dual assault on the tumor via delivery of asuper-cytotoxin combined with engagement of multiple arms of the immunesystem. This approach circumvents some of the current pitfalls withimmunotherapies by creating an immunogenic tumor microenvironment andalso acting on multiple immune cell subsets thereby avoiding primaryand/or adaptive resistances that may arise in patients.

Further, a subset of patients lack tumor immunogenicity resulting froman absence of tumor cell antigens or lack of immune cell infiltrationand therefore exhibit no initial response to the current strategiesavailable (Emens et al., 2017; Oiseth and Aziz, 2017; Sharma et al.,2017). Thus, the identification of novel, robust immunotherapeuticapproaches may result in significantly improved clinical outcomes andremains an area of high priority.

To mount an effective anti-tumor immune response, certain steps must beachieved either spontaneously or therapeutically. First, tumor cellantigens which may be derived in situ via tumor cell death, or deliveredexogenously must be taken up by dendritic cells (DC) (Anguille et al.,2015; Emens et al., 2017; Jung et al., 2018; Mellman et al., 2011). Inconjunction with antigen uptake, DCs need to receive a proper maturationsignal prompting differentiation and enhanced processing andpresentation of antigens such that antitumor function as opposed totolerance is promoted (Anguille et al., 2015; Emens et al., 2017; Junget al., 2018; Mellman et al., 2011; Simmons et al., 2012). These mature,tumor antigen loaded DCs must then effectively generate antitumor T-cellresponses which can occur via production of tumor specific cytotoxicT-cells, triggering of NK and/or NKT cell responses, and enhancingT-helper type 1 responses, among others (Emens et al., 2017; Fang etal., 2017; Mellman et al., 2011; Sharma et al., 2017; Zitvogel et al.,2015). Antitumor T-cells must finally enter the tumor microenvironment,where immune suppressive signals may be present, and effectively performtheir antitumor function (Emens et al., 2017; Mellman et al., 2011).Problems arising in any of these steps will impede efficacy of animmunotherapeutic, and can even result in total failure of the therapy(Emens et al., 2017; Mellman et al., 2011; Sharma et al., 2017).

Currently, the immunotherapeutic strategies which have received the mostattention clinically include immunological checkpoint inhibitors andchimeric antigen receptor T-cell therapy (CAR-T) (Emens et al., 2017;Mellman et al., 2011; Oiseth and Aziz, 2017; Sharma et al., 2017;Ventola, 2017). Checkpoint inhibitors such as cytotoxic T lymphocyteantigen 4 (CTLA-4), and programmed cell death 1/programmed cell death 1ligand (PD-1/PDL-1) function by blocking the transmission ofimmune-suppressive signals and direct stimulation to activate cytotoxicT lymphocytes within the tumor microenvironment (Dine et al., 2017;Jenkins et al., 2018; Sharpe, 2017). Inhibitors of these pathways haveshown dramatic clinical results in specific cancers, but overallresponse rates across different cancers remains low (˜15-25%) and immunerelated toxicities associated with these therapies can be high (Dine etal., 2017; Emens et al., 2017; Jenkins et al., 2018; Sharpe, 2017;Ventola, 2017). With new checkpoints continually being discovered aspotential immune targets, it is apparent that tumors are capable ofexploiting an elaborate and diverse set of immune-suppressive pathways(Dine et al., 2017; Emens et al., 2017; Farkona et al., 2016; Jenkins etal., 2018; Sharpe, 2017). Thus, development of resistance to checkpointinhibitors continues to be a hurdle and attempts are being made toutilize combinations of more than one checkpoint inhibitors to overcomethese issues, though this often exacerbates associated toxicities (Dineet al., 2017; Jenkins et al., 2018; Sharma et al., 2017; Ventola, 2017).

The second therapy receiving widespread attention is CAR-T cell therapywhich entails the genetic engineering of a patient's T-cells to expressmembrane fusion receptors with defined tumor antigen specificities andcapable of eliciting robust T-cell activation to initiate killing of thetarget tumor cells (D'Aloia et al., 2018′; Farkona et al., 2016; Mellmanet al., 2011; Sharma et al., 2017). This therapeutic approach hasproduced unprecedented clinical outcomes in the treatment of “liquid”hematologic cancers, but to date has not produced comparable responsesin targeting solid malignancies due to limitations associated with thelack of a good specific antigen target, poor homing to the tumor, poorextravasation into the tumor, and lack of persistence within a hostiletumor microenvironment (D'Aloia et al., 2018′; Sharma et al., 2017).Practical limitations relating to the availability of lymphocytes fromheavily pre-treated patients and long manufacturing times and are not afeasible treatment option for patients with rapidly advancing diseaseare also present (Oiseth and Aziz, 2017; Rezvani et al., 2017).

The EnGeneIC Dream Vector (EDV) is a bacterially-derived delivery systemconsisting of nonviable nanocells that are 400±20 nm in diameter,generated by reactivating polar sites of cell division in bacteria(MacDiarmid et al., 2007b). It has been demonstrated that thesenanocells can be packaged with a cytotoxic drug, siRNA, or miRNA andspecifically targeted to tumor cell-surface receptors via attachment ofbispecific antibodies to the surface polysaccharide of the nanocells(MacDiarmid et al., 2009; MacDiarmid et al., 2007b; Reid et al., 2013).Post-intravenous administration in mouse and dog studies hasdemonstrated that they are retained in the vascular system due to theirsize, but then rapidly extravasate into the tumor via thetumor-associated leaky vasculature (MacDiarmid et al., 2007b; Sagnellaet al., 2018). Post-tumor cell-surface receptor engagement via theassociated bispecific antibody results in macropinocytosis intoendosomes and release of the payload via degredation intracellularly inthe lysosomes (MacDiarmid et al., 2009; MacDiarmid et al., 2007b;Sagnella et al., 2018). The safety of these nanocell therapeutics hasbeen demonstrated in three Phase I clinical trials with over a thousanddoses administered in various end-stage cancer patients and 682 loadedEDVs are currently being delivered to patients in a phase I trial and todate have shown a promising safety profile (2017; Kao et al., 2015;Solomon et al., 2015; van Zandwijk et al., 2017; Whittle et al., 2015).

A. Overview of Bacterial Minicell Delivery Methods

The use of bacterial minicells to deliver chemotherapeutic agents tocancer cells has previously been described. This delivery method totreat cancer packages a toxic chemotherapy agent or drug, or functionalnucleic acid, into a bacterially-derived minicell, which are typicallyabout 400 nm in diameter. Typically, the minicell carries an antibodytargeting specific cancer cells. The antibodies attach to the surface ofcancer cells and the minicell is internalized by the cancer cell. Inthis way, the toxic chemotherapy agents are not widely distributedthroughout the body, and therefore reduce the chance of side effects andintolerability as the toxic drug or compound is delivered inside thecancer cell. Using antibody-targeted minicells as a delivery vehicle fortoxic chemotherapy agents results in much less drug needed to kill thecancer cell, thus improving the therapeutic index.

Indeed, the present inventors have shown that minicells (or EnGeneICDream Vehicles, EDVs) can deliver chemotherapy drugs, such as paclitaxelor doxorubicin, to xenograft tumors in mice (Example 1), dogs (Example2), and monkeys (Example 3). The targeted delivery ensures that thecancer cells receive most of the chemotherapeutic agent, resulting in alow level of toxicity. See Examples 1-3; see also MacDiarmid et al.,2007b; MacDiarmid et al., 2007a; MacDiarmid et al., 2009; and MacDiarmidet al., 2016. Furthermore, the minicells do not induce a significantimmune response in the xenograft models, and the minicells are welltolerated (Example 4). Thus, intact bacterially derived minicells are awell-tolerated vehicle for delivering anti-cancer drugs to patients,with examples including doxorubicin targeted to advanced solid tumors(Example 5), doxorubicin targeted to glioblastoma (Example 6), andMicroRNA-16a targeted to mesothelioma (Example 7).

These treatment strategies did not result in complete remission or cureof all cancers in all patients, however. Accordingly, there is a needfor improved cancer treatment therapies. The present inventorsdiscovered that using a combination of minicells having three differenttypes of payloads produced surprisingly dramatic and effective clinicalefficacy.

Specifically, the present inventors discovered that minicells comprisinga chemotherapy agent (in the examples below, for instance, the agent isPNU-159682, a supertoxic chemotherapy drug) combined with minicellscomprising an interferon type I agonist and/or an interferon type IIagonist resulted in synergistic anti-tumor effects and waswell-tolerated by a patient suffering from late stage pancreatic cancer.See Example 12. In fact, the late-stage pancreatic cancer patientexhibited markedly improved quality of life after this treatment, whichis remarkable for a patient at that stage. This triple or duelcombination strategy provides synergistically improved treatment ofcancers, particularly late-stage terminal cancer. The inventors alsodiscovered that minicells comprising a chemotherapy agent combined withminicells comprising an interferon type II agonist resulted insynergistic anti-tumor effects.

It was also surprisingly discovered that a dual combination of aminicell packaged antineoplastic agent, in combination with a type IIinterferon agonist, and in the absence of a type I interferon agonist,resulted in dramatic efficacy against large sized tumors. Such resultshave not previously been described. It is theorized that in somepatients, combining a type I interferon agonist and type II interferonagonist may be counterproductive, as the two types of interferonagonists may compete rather than synergistically act. This data isdescribed in more detailed below.

The following description outlines the invention related to thesediscoveries, without limiting the invention to the particularembodiments, methodology, protocols, or reagents described. Likewise,terminology used here describes particular embodiments only and does notlimit the scope of the invention.

B. Summary of the Experimental Results

(i) Minicell Packaged Antineoplastic Agent in Combination with MinicellPackaged Type I Interferon Agonist

In a first embodiment, described are compositions and methods relatingto a combination of a bacterial minicell packaged antineoplastic agentin combination with a type I interferon agonist packaged in a bacterialminicell.

Example 11 and FIG. 18 describe data showing the results in lung cancerxenograft models in mice treated with various minicell (EDVs)compositions, as summarized in the table below. The animals of Groups 1and 5 were administered a combination of a chemotherapeutic agent (PNU159682) packaged in an intact bacterially derived minicell and a type Iinterferon agonist (a 40mer double stranded DNA or a 50merdouble-stranded DNA), also packaged in an intact bacterially derivedminicell. All of the minicell compositions resulted in stabilization oftumor growth. However, the most dramatic results were obtained aftertreating the large tumor size that resulted from saline treatment inpart 1 of the experiment. When the saline-treated control group wassubsequently treated in part 2 of the experiment with a compositioncomprising a combination of minicell-packaged anti-neoplastic agent plusa minicell-packaged type I interferon agonist, the tumor size wasreduced by 62% over a 5-day period.

TABLE 1 Phase II Treatment Phase I Starting at days Group Treatment FIG.Results 36 and 38 Results 1 ^(EGFR)EDV_(S) _(PNU-159682) + FIG. 18,solid Tumor growth EDV_(S) _(40mer) triangle stabilization 2^(EGFR)EDV_(S) _(PNU-159682) FIG. 18, solid Tumor growth circlestabilization 3 ^(EGFR)EDV_(S) _(PNU-159682) + FIG. 18, open Tumorgrowth EDVs square stabilization (no payload) 4 ^(EGFR)EDV_(S)_(PNU-159682) + FIG. 18, open Tumor growth EDV_(S) _(50mer) trianglestabilization 5 Saline FIG. 18, solid tumor growth up Treatment with In5 days, tumors square to a volume of ^(EGFR)EDV_(S) _(PNU-159682) +having a large ~650 mm³ EDV_(S) _(40mer) volume of ~650 mm³ decreased to~250 mm³- or a 62% reduction in size in 5 days

In a follow-up of Example 11 (results shown in FIG. 19), the addition ofa type I interferon agonist packaged in a minicell resulted in dramatictumor size reduction, which was not seen when an anti-neoplastic agentpackaged in a minicell was used in the absence of the type I interferonagonist adjuvant. The results are summarized in the table below. Theseresults clearly demonstrate the adjuvant effects on minicell-packagedanti-neoplastic agents with the addition of a minicell-packaged type Iinterferon agonist.

TABLE 2 Group Treatment FIG. Results 1 ^(EGFR)EDV_(S) _(PNU-159682) FIG.19, solid circle Slight tumor size reduction (from a tumor volume ofabout 275 mm³ to 260 mm³) 2 solid triangle = FIG. 19, solid triangleSignificant tumor reduction, from a tumor ^(EGFR)EDV_(S) _(PNU-159682) +volume about 275 mm³ to about 175 mm³) EDV_(S) _(40mer) 3 Saline FIG.19, solid square Significant tumor growth.

(ii) Minicell Packaged Antineoplastic Agent in Combination with MinicellPackaged Type I Interferon Agonist and Optionally a Type II InterferonAgonist (not Minicell Packaged)

A second embodiment is directed to methods and compositions utilizing aminicell packaged antineoplastic agent combined with a mincell-packagedtype I interferon agonist or a minicell-packaged anti-neoplastic agentin combination with a mincell-packaged type I interferon agonist, and atype II interferon agonist (free from a bacterial minicell).

Further evidence of the dramatic and surprising effectiveness of thecompositions of the invention is reflected in the clinical results shownin Example 12. Specifically, Example 12 relates what happened whenpatients suffering from advanced solid tumors were treated withcompositions comprising (1) a combination of a minicell-packagedanti-neoplastic agent and a mincell-packaged type I interferon agonist;and (2) a combination of a minicell-packaged anti-neoplastic agent, amincell-packaged type I interferon agonist, and a type II interferonagonist.

In particular, the human clinical data detailed in Example 12demonstrate the safety profile of type I and type II IFN agonists usedas adjuvants for minicell-packaged anti-neoplastic agents in humanpatients. See data in Table 3 below, in relation to which the type Iinterferon agonist packaged in intact, bacterially-derived minicells was40mer double-stranded DNA (EDVs_(40mer)) or 60mer double-stranded DNA(EDVs_(60mer)).

Moreover, the results obtained with a stage 4 pancreatic cancer patient,who had exhausted all other treatment options, were remarkable. Thelevels of the patient's tumor marker (CA 19-9) dropped by more than 90%after the initial three doses, equivalent to only 10 days of treatment.After ten doses this had dropped even further, with an almost 95%reduction in tumor marker levels. The patient also demonstratedsignificant weight gain, in contrast to the cachexic state experiencedby most patients presenting with stage IV pancreatic cancer, andreported a marked improvement in quality of life. These results aredramatic, particularly given the poor prognosis associated with advancedpancreatic cancer.

In summary, five patients received a total of 69 doses of^(EGFR(V))EDVs_(PNU/Dox) or ^(EGFR(V))EDVs_(PNU)+EDVs_(40mer/60mer),(type I IFN agonist)±Imukin (type II IFN agonist). The treatments werewell-tolerated, and the addition of immunomodulatory adjuvants did notseem to change the safety profile of single-agent-loaded and targetedEDVs.

TABLE 3 Patient # Treatment Cancer Comments 3 patients^(EGFR(V))EDVs_(PNU-159682) advanced solid Treatment was well tolerated,no at 2.5 × 10⁹ + EDVs_(40mer) tumors unexpected adverse reactions. Oneat 5 × 10⁸ patient was ultimately withdrawn from the study due todose-limiting toxicity. 1 patient ^(EGFR(V))EDVs_(PNU-159682) Stage IVpancreatic Treatment was well-tolerated; levels and EDVs_(40mer) orcancer of the patient's tumor marker (CA 19- EDVs_(60mer) And 9) droppedby more than 90% after ITG-targctcd EDVs loaded the first 3 doses,equivalent to only 10 with PNU-159682 days of treatment. After 10 dosesthis (^(ITG(609))EDVs_(PNU)) had dropped even further, with an almost95% reduction in tumor marker levels. 1 patient ^(EGFR(V))EDVs_(PNU)recurrent and end- Treatment was well tolerated. and EDVs_(60mer) +Imukin stage adreno-cortical (type II IFN agonist) cancer with a veryheavy tumor burden

(iii) Minicell Packaged Antineoplastic Agent in Combination with Type IIInterferon Agonist

Example 13 describes the results of various studies conducted toevaluate the effectiveness of combining anti-neoplastic agents packagedin minicells with a type II interferon agonist, e.g., IFN-γ. The resultsshow that the addition of a type II interferon agonist augments orenhances the anticancer effect of anti-neoplastic agents packaged inminicells in xenograft models of various cancers, including lung cancerand breast cancer. Further, the data set forth in Example 13 andexcerpted in Table 4 below demonstrate that the addition of a type IIinterferon agonist to a composition comprising an antineoplastic agentpackaged in a minicell in the treatment of tumors normally resistant tothe antineoplastic agent alone is essential to achieve tumorstabilization. Thus, combining a minicell-packaged antineoplastic agentwith a type II interferon agonist can overcome drug resistance.

TABLE 4 Cancer Treatment FIG. Results Lung Group 1 = sterile FIG. 20,open diamonds Significant tumor growth cancer physiological saline Group2 = IFN-γ (0.5 × 10⁴ FIG. 20, solid triangles no anti-tumor efficacy IU)per dose Group 3 = ^(EGFR)EDVs_(Dox) FIG. 20, solid squares tumorstabilisation Group 4 = ^(EGFR)EDVs_(Dox) and FIG. 20, solid circleshighly significant tumor IFN-γ (0.5 × 10⁴ IU) per dose regression by day43 after a total of 6 doses Breast Group 1 = sterile FIG. 21, opendiamonds Significant tumor growth cancer physiological saline Group 2 =IFN-γ (0.5 × 10⁴ FIG. 21, solid triangles no anti-tumor efficacy IU)Group 3 = ^(EGFR)EDVs_(Dox) FIG. 21, solid squares tumor stabilisationof breast cancer xenografts, but by ~day 25 the tumors began to growagain, likely due to development of resistance to doxorubicin Group 4 =^(EGFR)EDVs_(Dox) and FIG. 21, solid circles highly significant tumorIFN-γ (0.5 × 10⁴ IU) per dose regression, and by day 30, after a totalof 6 doses, these tumors were more like scar tissue

(iv) Triple Combination of a Minicell-Packaged Antineoplastic Agent, aMinicell-Packaged Type I Interferon Agonist, and a Type II InterferonAgonist (Either Alone or Minicell-Packaged)

The present inventors also discovered that a triple combination of aminicell-packaged antineoplastic agent, a minicell-packaged type Iinterferon agonist, and a type II interferon agonist (either alone orminicell-packaged) can produce dramatic anticancer effects.Specifically, Example 14 details treatment of dogs with late stageendogenous tumors (brain cancer, sarcoma, or melanoma) with acombination of a minicell-packaged antineoplastic agent, aminicell-packaged type I interferon agonist, and a type II interferonagonist. The results show that the combination composition waswell-tolerated. Moreover, in 6 of 7 evaluable animals (85.7%) thedisease was stabilized, although one dog achieved a near partialresponse (29.8% reduction in tumor size).

(v) Duel Combination of a Minicell-Packaged Antineoplastic Agent inCombination with a Minicell-Packaged Type II Interferon Agonist, in theAbsence of a Type I Interferon Agonist

In another embodiment, this invention relates to the surprisingdiscovery that compositions comprising a combination of aminicell-packaged antineoplastic agent and a minicell packaged type IIinterferon agonist, such as for example alpha-galactosyl ceramide(α-GC), and in the absence of a type I interferon agonist, demonstratessurprising anticancer efficacy.

In particular, Example 23 describes data illustrating the efficacy of adual combination of minicell contained therapeutic and minicellcontained interferon type II agonist against tumors. This resultdemonstrates that compositions lacking interferon type I agonists can beused to effectively treat tumors. See also, FIGS. 40 and 42. Theexperimental results showed a marked halt in tumor progression forcombination treatment groups receiving ^(Ep)minicell_(Dox)+minicell_(α-GC) (interferon type II agonist) as compared to saline and^(Ep)minicell_(Dox) treatments. This result supports the theory of animmune adjuvant effect by the addition of minicell_(α-GC) treatment to^(Ep)minicell_(Dox).

Further data showed that saline treated control tumors demonstrateddramatic tumor regression following a treatment change to drug and α-GCEDV mediated dual combination therapy (FIG. 41); e.g., a combination ofminicell packaged antineoplastic agent and minicell packaged type IIinterferon agonist. In particular, tumours that had reached 800 mm³dropped to below 600 mm³ in 3 days before the experiment wasterminated—a markedly dramatic tumor size reduction (˜25%) in a shortperiod of time. The ability for the dual combination composition todramatically decrease large tumors in a short period of time was notknown prior to the present invention.

In one embodiment of the invention, the dual combination composition(e.g., a minicell packaged antineoplastic agent in combination with aminicell packaged interferon type II agonist) can reduce a tumour'ssize, including the size of a large tumor, by about 5%, about 10%, about15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%,about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90%, about 95%, or about 100%. The reduction intumor size can be measured over any suitable time period, such as about3 days, about 5 days, about 1 week, about 2 weeks, about 3 weeks, about1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about9, about 10, about 11, about 12 months, about 1.5 years, about 2 yearsor longer.

C. Immunotherapy Data

Example 16 details data demonstrating that minicell packagedantineoplastic agents targeted to a tumor cell surface receptor functionas a cancer immunotherapy, e.g., as a cyto-immunotherapy. In particular,the example illustrates the ability of the bacterial minicell toactivate cells of the innate immune system, including macrophages, NKcells and dendritic cells. This is followed by dendritic cell maturationand antigen presentation leading to an adaptive T-cell response in whichtumor specific cytotoxic T-cells are produced and results in furtherrecruitment of additional immune cells to the tumor microenvironment.This approach circumvents some of the current pitfalls withimmunotherapies by creating an immunogenic tumor microenvironment andalso acting on multiple immune cell subsets thereby avoiding primaryand/or adaptive resistances that may arise in patients.

This example therefore shows the ability of the bacterial minicell todeliver a cytotoxic drug within tumor cells and to also simultaneouslyelicit an innate and adaptive immune response specifically targeting thetumor.

Further immunotherapy data is shown in Example 18, which describes datashowing that NK cells adopt an antitumor phenotype in vivo followingtreatment with targeted minicells comprising an antineoplastic agent.This is significant as NK cells are the primary effector cell of theinnate immune system and are tightly regulated by a balance ofactivating and inhibitory signals (Morvan and Lanier, 2016; Wallace andSmyth, 2005). Impairment of NK cell function has been associated withincreased tumor incidence, growth, and metastasis, and thus itsimportance in contributing to an antitumor immune response is welldocumented (Fang et al., 2017; Morvan and Lanier, 2016; Rezvani et al.,2017; Wallace and Smyth, 2005).

Interesting, Example 19 details data showing that a predominantly Th1cytokine response within a tumor microenvironment is exhibited followingtreatment with a minicell-encapsulated antineoplastic agent (e.g.,PNU-159682). Cytokine and chemokine production within a tumormicroenvironment allows immune cells to effectively communicate witheach other to generate a coordinated response which can either be tumorpromoting or suppressing (Belardelli and Ferrantini, 2002; Lee andMargolin, 2011). The effect of individual cytokines on immune responseis dependent on a variety of factors including local concentration,cytokine receptor expression patterns and the activation state ofsurrounding cells (Lee and Margolin, 2011). Thus, many cytokines havebeen shown to be capable of eliciting opposing effects on tumor growth(Dredge et al., 2002; Landskron et al., 2014; Lee and Margolin, 2011).

Further, Example 20 details data showing that treatment with aminicell-encapsulated antineoplastic agent (e.g., PNU-159682) results inthe production of tumor specific CD8+ T-cells. Initial in vitroexperiments indicated that EDV treatment can result in dendritic cellmaturation either via direct interaction or as a result of cell death inresponse to a targeted EDV loaded with an effective chemotherapeutic.Thus, this experiment aimed to examine if this result could translate toDC maturation and antigen presentation in vivo resulting in theproduction of tumor specific CD8⁺ cytotoxic T-cells. The resulting datademonstrated that minicell treatment successfully elicited theproduction of tumor specific CD8⁺ T-cells. In addition, a significantincrease in overall T-cell numbers (CD3+) as well as a significantincrease in both CD4+ and CD8+ T-cells numbers were seen in the lymphnodes of mice treated with minicell encapsulated antineoplastic agent(e.g., PNU-159682) (FIG. 31G). A significant increase in maturedendritic cells in the lymph nodes of treated mice was also detected(FIG. 31H), and visualization of the interaction between isolated CD8⁺T-cells from treated mice with 4T1 cells shows that these T-cells arecapable of attaching to and expelling perforin (green) into the tumorcell (FIG. 31I).

Example 21 demonstrates the ability of targeted bacterial minicellsloaded with an antineoplastic agent (e.g., the super-cytotoxinPNU-159682) to not only effectively deliver this drug to the tumor site,but also behave as an immunotherapeutic by stimulating multiple immunecell subsets. The example demonstrates the ability of a minicellcapsulated antineoplastic agent treatment to push immune cell subsets,including macrophages, NK cells and CD8⁺ T-cells, towards an antitumorphenotype capable of effectively eliminating tumor cells. When combinedwith the effectiveness of an antineoplastic agent, this results in adual assault on the tumor.

While the idea of cancer immunotherapy has been around for decades, itis only in recent times that its potential has begun to be realized withthe approval of a number of immunotherapies (Farkona et al., 2016;Ventola, 2017). Bacterial minicells represent a unique, combinedcyto-immunotherapy which first creates an immunogenic tumormicroenvironment via the delivery of cytotoxic agents directly to thetumor, where it stimulates the innate immune system either directly orindirectly towards an antitumor phenotype. This innate immune activationthen triggers an adaptive response in which tumor specific cytotoxicT-cells arise (FIG. 33).

Following intravenous administration, the minicell extravasates to atumor via the tumor's leaky vasculature where ≥30% of the administereddose of targeted minicells carrying their toxic payload deposit directlyinto the tumor microenvironment within a 2 hr period (MacDiarmid et al.,2007b). Targeted bacterial minicells bind to receptors on the tumorcells (4T1 and CT26Ep12.1 in the case of Example 21), and are theninternalized effectively delivering their payload (antineoplastic agent)directly within the tumor cells. PNU-159682 is a highly potent supercytotoxin resulting in rapid apoptosis within 24h of being delivered tothe tumor cells (FIG. 33A). The apoptotic cells and DAMP signalsproduced by bacterial minicell (e.g., Ep-EDV-682) treatment can theninteract with innate immune cells such as tumor associated macrophages(TAMs) and stimulate upregulation of CD86 and the production of Th1pro-inflammatory cytokines such as TNFα and IL-6 (FIG. 33B). Thesechanges are typical of M1 polarization of macrophages which are capableof lysing tumor cells and releasing cytokines to signal activation ofother immune cell subsets, and have thus been shown to possess antitumorcharacteristics (Sawa-Wejksza and Kandefer-Szerszen, 2018; Yuan et al.,2015).

Furthermore, the bacterial minicell itself can also interact directlywith TAMs producing a similar M1 polarization, albeit this would beexpected to occur at very low levels in the current system. TAMs aregenerally the most abundant immune cell in the tumor microenvironment,and it has been demonstrated that increased numbers of TAMs areassociated with poor prognosis and increased tumor growth (Sawa-Wejkszaand Kandefer-Szerszen, 2018). This is due in large part to the fact thatTAMs mostly consist of anti-inflammatory M2 macrophages which have beenshown to possess tumor promoting characteristics, whereas inflammatoryM1 macrophages exhibit antitumor characteristics (Sawa-Wejksza andKandefer-Szerszen, 2018; Yuan et al., 2015). Example 21 demonstrates theability of bacterial minicell treatment to shift the M1:M2 balancewithin the tumor microenvironment in 4 different tumor models. Despitedifferences in the degree of this shift in the different tumor models,it was shown that the increase in M1 polarization translated toincreased tumor cell lysis by TAMs isolated from the tumors of micewhich had been treated with bacterial minicells. In addition to thephenotypic shifts to M1, TAMs from tumors of bacterial minicell treatedmice also secreted an increased amount of MIP-1 (FIG. 33C), a chemokinewhich has been established to play a role in promoting immune cellrecruitment, and in particular tumor infiltration by NK cells, CD4⁺T-cells and CD8⁺ T-cells (Allen et al., 2018).

In addition to TAM activation, immature dendritic cells (DC) interacteither directly with bacterial minicells, or more likely, with theapoptotic cells and DAMP signals produced by bacterial minicell treatedtumors resulting in dendritic cell maturation and migration to the lymphnodes for antigen presentation. DCs have been explored as a potentialtarget in cancer immunotherapies as they are known to be the mosteffective antigen presenting cell and constitute the bridge between theinnate and adaptive immune system (Allen et al., 2018).

Most current strategies for DC based immunotherapy involve ex vivomanipulation and priming of DCs or DC precursors, however success fromthis strategy has been limited due to a variety of factors including:development of immune tolerance, induction of insufficient numbers ofCD8+ cytotoxic T-cells (CTL) or those with poor antitumor efficacy, andthe suppressive nature of the tumor microenvironment (Anguille et al.,2015; Jung et al., 2018; Landskron et al., 2014; Oiseth and Aziz, 2017).Bacterial minicell treatment allows for in vivo priming and maturationof DCs within the tumor microenvironment in response to dying tumorcells (FIG. 33D). Immature DCs are capable of engulfing DAMPs and/orapoptotic tumor cell bodies produced in response to targeted, drugloaded bacterial minicells. These DAMPs and dying tumor cells are thenprocessed for antigen presentation on the DC surface via MHC Class I andII molecules, with concomitant DC maturation. Upregulation of theco-stimulatory molecules CD86, CD80 and MHC Class II, which have beenidentified as markers of the DC maturation process, was shown to occurin DCs co-cultured with bacterial minicell treated tumor cells, alongwith an increase in the percentage of mature DCs detected in the tumordraining lymph nodes of bacterial minicell treated mice (Anguille etal., 2015; Cauwels et al., 2018; Simmons et al., 2012). During thematuration process, the DCs migrate to the tumor draining lymph nodesfor antigen presentation to T-cells thereby increasing production ofCD4⁺ T-helper cells and tumor specific CD8⁺ CTL initiating an adaptiveimmune response to the tumor (FIG. 33E). An increase in the productionof IFNα/β, TNFα, IL-12p40, and IL-6 by DCs co-cultured with bacterialminicell treated tumor cells was subsequently detected, in addition to asignificant increase in IFNα concentration in the tumor microenvironmentobserved in both the 4T1 and CT26Ep12.1 tumor models (Example 21).Expression levels of type 1 IFNs (IFNα/β) and IFN stimulated geneswithin the tumor microenvironment have been shown to correlate withfavorable disease outcomes and may in fact even be necessary for thesuccess of cancer therapies including immunotherapies (Cauwels et al.,2018; Fitzgerald-Bocarsly and Feng, 2007; Zitvogel et al., 2015). Theantitumor activity of type 1 IFNs arise indirectly via immune cellactivation of DCs, T and B lymphocytes, NK cells, and macrophages(Cauwels et al., 2018; Fitzgerald-Bocarsly and Feng, 2007; Showalter etal., 2017; Zitvogel et al., 2015).

In conjunction with enhancing macrophage and DC antitumor functions,treatment with bacterial minicells comprising an antineoplastic agent iscapable of eliciting NK cell activation leading to increasedcytotoxicity (FIG. 33F). NK cells possess the inherent ability to lysemalignant cells in an antigen independent manner, thus their activationand functional status must be tightly controlled in order to avoidpotentially adverse effects on the host. The ability to attract NK cellsto and activate NK cells within the tumor microenvironment is vital totheir ability to exert their antitumor function. Cytokines includingIL-2, IFNγ, and IFNα which are significantly increased in themicroenvironment of Ep-EDV-682 treated tumors, are known to activate NKcells towards both increased cytokine production and enhanced cytolyticfunction (Fang et al., 2017; Ferlazzo and Munz, 2004; Lee and Margolin,2011; Morvan and Lanier, 2016; Rezvani et al., 2017). In fact, evidenceindicates that type 1 IFNs are required for the activation of NK cellcytotoxicity (Ferlazzo and Munz, 2004; Muller et al., 2017). Further,type 1 IFNs are capable of inducing cellular senescence followed byupregulation of NKG2D ligand expression in tumor cells thereby promotingtheir elimination by NK cells (Muller et al., 2017). Upregulation of theNKG2D receptor was observed on NK cells within the tumors of micetreated with Ep-EDV-682, and this receptor was demonstrated tocontribute significantly to the cytolytic ability of NK cells isolatedfrom Ep-EDV-682 treated mice. Moreover, immature, intermediate andmature mouse NK cells express both the CCR1 and CCR5 chemokine receptorsthat can bind the chemokines MIP1α and RANTES, both of which areupregulated in Ep-EDV-682 treated tumors as well as by macrophages andNK cells from Ep-EDV-682 treated mice (Bernardini et al., 2016).

Chemokines, such as MIP1α and RANTES, are responsible for the furtherrecruitment of helper and effector immune cells including NK cells,macrophages, and T-cells to the tumor microenvironment (FIG. 33G) (Allenet al., 2018; Bernardini et al., 2016; Zibert et al., 2004). Followingthe initial innate immune response due to EDV treatment whichencompasses macrophages, NK cells, and DCs, an adaptive immune responseis mounted in which tumor specific CTLs and T-helper cells are producedand then recruited to the tumor site (FIG. 33H). Tumor specific CTLsthen target and lyse tumor cells further contributing to the overridingantitumor environment which has been created by the other immune cellsubsets in combination with the targeted, drug loaded EDVs. Targeted,drug loaded EDV treatment elicits a mainly Th1 response as evidenced bythe increase of Th1 cytokines (TNFα, IFNα, IFNγ, IL-2, and IL-6) withinthe tumor microenvironment. As previously mentioned, innate immune cellsubsets, when activated, become a primary source of one or more of theseparticular cytokines. T-cells are similarly capable of producing all ofthe aforementioned cytokines (Belardelli and Ferrantini, 2002; Lee andMargolin, 2011). Release of these cytokines by either innate immunecells or T-cells are responsible for co-stimulation, activation, growth,and increased antigen presentation of additional immune cells creating afeedback loop which further enhances the antitumor activity of theimmune system FIG. 33I) (Lee and Margolin, 2011).

Bacterial minicell treatment represents a unique cancer therapeuticstrategy capable of delivering conventional and novel drug therapiesdirectly to the tumor site and subsequently eliciting an antitumorimmune response. A dual assault on the tumor occurs, first through celldeath in response to the delivered therapeutic and followed by innateimmune cell activation leading to an adaptive immune response. This typeof therapy has certain advantages over current immunotherapy strategiesin that immune cell activation occurs both in vivo and primarily at thetumor site, which is a rapidly changing, dynamic environment. Further,it creates an immunogenic tumor environment and elicits effects onmultiple immune cell subsets avoiding problems associated with patientswho show little to no immune response to their tumors or adaptations totherapies which only target single immune cell subsets. The studydescribed in Example 21 highlights the potential of bacterial minicellsas a novel cancer immunotherapeutic, and future bacterial minicellformulations could further exploit its inherent immunogenic nature giventhe versatility of this technology with respect to both payload andtargeting ability (MacDiarmid et al., 2007a).

D. Supertoxic Antineoplastic Agents

Example 17 details data demonstrating the effective delivery of a supertoxic antineoplastic agent, e.g., PNU-159682, which is unable to bedelivered using conventional means because of severe toxicity associatedwith the compound. Specifically, Example 17 details how PNU-159682 is asuper cytotoxin with IC50s for even drug-resistant cancer cells in thepM range (Quintieri et al., 2005), which means that the compound isunable to be used clinically due to the severe systemic toxicity(Staudacher and Brown, 2017). However, when encapsulated in a bacterialminicell, super cytotoxins such as PNU-159682 can be effectivelydelivered to the tumor with few side effects.

II. Composition Components

As noted above, the compositions of the invention comprise at least twodifferent active agents, an antineoplastic agent and a type I interferonagonist, a type II interferon agonist, or both a type I interferonagonist and a type II interferon agonist with the antineoplastic agent.The three different active agents can be packaged in one, two, or threedifferent minicells. The type II interferon agonist also can be includedin the methods and compositions of the invention without being packagedin a minicell.

A. Antineoplastic or Cytotoxic Active Agents Useful in Treating Cancer

The phrase “anti-neoplastic agent” denotes a drug, whether chemical orbiological, that prevents or inhibits the growth, development,maturation, or spread of neoplastic cells. The term “antineoplasticagent” is used interchangeably with “anticancer agent” and “chemotherapyagent.”

In the context of this disclosure, selecting an anti-neoplastic agentfor treating a given brain tumor patient depends on several factors, inkeeping with conventional medical practice. These factors include butare not limited to the patient's age, Karnofsky Score, and whateverprevious therapy the patient may have received. See, generally,PRINCIPLES AND PRACTICE OF NEURO-ONCOLOGY, M. Mehta (Demos MedicalPublishing 2011), and PRINCIPLES OF NEURO-ONCOLOGY, D. Schiff and P.O'Neill, eds. (McGraw-Hill 2005).

The composition can comprise at most about 1 mg of the antineoplastic orchemotherapeutic drug. Alternatively, the amount of the chemotherapeuticdrug can be at most about 750 μg, about 500 μg, about 250 μg, about 100μg, about 50 μg, about 10 μg, about 5 μg, about 1 μg, about 0.5 μg, orabout 0.1 μg. In another aspect, the composition comprises achemotherapeutic drug having an amount of less than about 1/1,000, oralternatively less than about 1/2,000, 1/5,000, 1/10,000, 1/20,000,1/50,000, 1/100,000, 1/200,000 or 1/500,000 of the therapeuticallyeffective amount of the drug when used without being packaged intominicells. Pursuant to yet another aspect of the disclosure, thecomposition can comprise at least about 1 nmol of the chemotherapeuticdrug. Accordingly, the disclosure also encompasses embodiments where theamount of the chemotherapeutic drug is at least about 2 nmol, about 3nmol, about 4 nmol, about 5 nmol, about 10 nmol, about 20 nmol, about 50nmol, about 100 nmol, or about 800 nmol, respectively.

In the context of this disclosure, selecting an anti-neoplastic agentfor treating a given tumor depends on several factors. These factorsinclude but are not limited to the patient's age, the stage of thetumor, and whatever previous therapy the patient may have received.

In accordance with the disclosure, a drug can be selected from one ofthe classes detailed below for packaging into intact, bacteriallyderived minicells. These drugs can also be synthetic analogs designedfrom drug design and discovery efforts. Any known chemotherapy agent canbe utilized in the compositions of the invention. Examples of knownchemotherapy agents include, but are not limited to:

(1) alkylating agents, such as mustard gas derivatives (Mechlorethamine,Cyclophosphamide (Cytoxan), Chlorambucil (Leukeran), Melphalan, andIfosfamide), ethylenimines (Thiotepa (Thioplex) and Hexamethylmelamine),alkylsulfonates (Busulfan (Myleran)), hydrazines and triazines(Altretamine (Hexalen), Procarbazine (Matulane), Dacarbazine (DTIC) andTemozolomide), nitrosureas (Carmustine, Lomustine and Streptozocin), andmetal salts (Carboplatin, Cisplatin (Platinol), and Oxaliplatin),Mechlorethamine, and Melphalan (Alkeran);

(2) Plant alkaloids, terpenoids and topoisomerase inhibitors, such asvinca alkaloids (Vincristine (Oncovin), Vinblastine (Velban), Vindesine,and Vinorelbine), taxanes (Paclitaxel (Taxol) and Docetaxel (Taxotere)),podophyllotoxins (Etoposide and Tenisopide), and camptothecan analogs(Irinotecan and Topotecan);

(3) antitumor antibiotics, such as anthracyclines (Doxorubicin(Adriamycin, Rubex, Doxil), Daunorubicin, Epirubicin, Mitoxantrone,Idarubicin, Duocarmycin, and Dactinomycin (Cosmegen)), chromomycins(Dactinomycin and Plicamycin (Mithramycin)), and miscellaneous(Mitomycin and Bleomycin (Blenoxane));

(4) antimetabolites, such as folic acid antagonists (Methotrexate),pyrimidine antagonists (5-Fluorouracil, Foxuridine, Cytarabine,Flurouracil (5-FU), Capecitabine, and Gemcitabine), purine antagonists(6-Mercaptopurine (Purinethol) and 6-Thioguanine), 6-Thiopurines, andadenosine deaminase inhibitor (Cladribine (Leustatin), Fludarabine,Nelarabine and Pentostatin), Azacitidine, Thioguanine, and Cytarabine(ara-C);

(5) topoisomerase Inhibitors, such as topoisomerase I inhibitors(Ironotecan, topotecan), and topoisomerase II inhibitors (Amsacrine,etoposide, etoposide phosphate, teniposide);

(6) hormonal agents, exemplified by Estrogen and Androgen Inhibitors(Tamoxifen and Flutamide), Gonadotropin-Releasing Hormone Agonists(Leuprolide and Goserelin (Zoladex)), Aromatase Inhibitors(Aminoglutethimide and Anastrozole (Arimidex));

(7) DNA hypomethylating agents, e.g., Azacitidine, Decitabine;

(8) Poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) pathwayinhibitors, such as Iniparib, Olaparib, Veliparib;

(9) PI3K/Akt/mTOR pathway inhibitors, e.g., Everolimus;

(10) Histone deacetylase (HDAC) inhibitors, e.g., Vorinostat, Entinostat(SNDX-275), Mocetinostat (MGCD0103), Panobinostat (LBH589), Romidepsin,Valproic acid.

Cyclin-dependent kinase (CDK) inhibitors, e.g., Flavopiridol,Olomoucine, Roscovitine, Kenpaullone, AG-024322 (Pfizer), Fascaplysin,Ryuvidine, Purvalanol A, NU2058, BML-259, SU 9516, PD-0332991, P276-00.[0050] Heat shock protein (HSP90) inhibitors, e.g., Geldanamycin,Tanespimycin, Alvespimycin, Radicicol, Deguelin, and BIIB021;

(11) Murine double minute 2 (MDM2) inhibitors, e.g., Cis-imidazoline,Benzodiazepinedione, Spiro-oxindoles, Isoquinolinone, Thiophene,5-Deazaflavin, Tryptamine;

(12) Anaplastic lymphoma kinase (ALK) inhibitors, e.g., Aminopyridine,Diaminopyrimidine, Pyridoisoquinoline, Pyrrolopyrazole, Indolocarbazole,Pyrrolopyrimidine, Dianilinopyrimidine;

(13) Poly [ADPribose] polymerase (PARP) inhibitors, illustrated byBenzamide, Phthalazinone, Tricyclic indole, Benzimidazole, Indazole,Pyrrolocarbazole, Phthalazinone, Isoindolinone; and

(14) miscellaneous anticancer drugs, exemplified by Amsacrine,Asparaginase (El-spar), Hydroxyurea, Mitoxantrone (Novantrone), Mitotane(Lysodren), Maytansinoid, Retinoic acid Derivatives, Bone Marrow GrowthFactors (sargramostim and filgrastim), Amifostine, agents disruptingfolate metabolism, e.g., Pemetrexed, ribonucleotide reductase inhibitors(Hydroxyurea), adrenocortical steroid inhibitors (Mitotane), enzymes(Asparaginase and Pegaspargase), antimicrotubule agents (Estramustine),and retinoids (Bexarotene, Isotretinoin, Tretinoin (ATRA)).

Chemotherapy drugs that are illustrative of the small molecule drugsubcategory are Actinomycin-D, Alkeran, Ara-C, Anastrozole, BiCNU,Bicalutamide, Bleomycin, Busulfan, Capecitabine, Carboplatin,Carboplatinum, Carmustine, CCNU, Chlorambucil, Cisplatin, Cladribine,CPT-11, Cyclophosphamide, Cytarabine, Cytosine arabinoside, Cytoxan,Dacarbazine, Dactinomycin, Daunorubicin, Dexrazoxane, Docetaxel,Doxorubicin, DTIC, Epirubicin, Ethyleneimine, Etoposide, Floxuridine,Fludarabine, Fluorouracil, Flutamide, Fotemustine, Gemcitabine,Hexamethylamine, Hydroxyurea, Idarubicin, Ifosfamide, Irinotecan,Lomustine, Mechlorethamine, Melphalan, Mercaptopurine, Methotrexate,Mitomycin, Mitotane, Mitoxantrone, Oxaliplatin, Paclitaxel, Pamidronate,Pentostatin, Plicamycin, Procarbazine, Steroids, Streptozocin, STI-571,Streptozocin, Tamoxifen, Temozolomide, Teniposide, Tetrazine,Thioguanine, Thiotepa, Tomudex, Topotecan, Treosulphan, Trimetrexate,Vinblastine, Vincristine, Vindesine, Vinorelbine, VP-16, and Xeloda.

Maytansinoids (molecular weight: ˜738 Daltons) are a group of chemicalderivatives of maytansine, having potent cytotoxicity. Althoughconsidered unsafe for human patient use, due to toxicity concerns,maytansinoids are suitable for delivery to brain tumor patients viaminicells, pursuant to the present invention.

Duocarmycins (molecular weight: ˜588 Daltons) are a series of relatednatural products, first isolated from Streptomyces bacteria. They alsohave potent cytotoxicity but are considered as unsafe for human use.Like maytansinoids, duocarmycins are suitable chemotherapy drugs for usein the invention.

The subcategory of biologic chemotherapy drugs includes, withoutlimitation, Asparaginase, AIN-457, Bapineuzumab, Belimumab, Brentuximab,Briakinumab, Canakinumab, Cetuximab, Dalotuzumab, Denosumab,Epratuzumab, Estafenatox, Farletuzumab, Figitumumab, Galiximab,Gemtuzumab, Girentuximab (WX-G250), Herceptin, Ibritumomab, Inotuzumab,Ipilimumab, Mepolizumab, Muromonab-CD3, Naptumomab, Necitumumab,Nimotuzumab, Ocrelizumab, Ofatumumab, Otelixizumab, Ozogamicin,Pagibaximab, Panitumumab, Pertuzumab, Ramucirumab, Reslizumab,Rituximab, REGN88, Solanezumab, Tanezumab, Teplizumab, Tiuxetan,Tositumomab, Trastuzumab, Tremelimumab, Vedolizumab, Zalutumumab, andZanolimumab.

In some embodiments, the anti-neoplastic agent comprises a compoundselected from the group consisting of actinomycin-D, alkeran, ara-C,anastrozole, BiCNU, bicalutamide, bleomycin, busulfan, capecitabine,carboplatin, carboplatinum, carmustine, CCNU, chlorambucil, cisplatin,cladribine, CPT-11, cyclophosphamide, cytarabine, cytosine arabinoside,cytoxan, dacarbazine, dactinomycin, daunorubicin, dexrazoxane,docetaxel, doxorubicin, DTIC, epirubicin, ethyleneimine, etoposide,floxuridine, fludarabine, fluorouracil, flutamide, fotemustine,gemcitabine, hexamethylamine, hydroxyurea, idarubicin, ifosfamide,irinotecan, lomustine, mechlorethamine, melphalan, mercaptopurine,methotrexate, mitomycin, mitotane, mitoxantrone, oxaliplatin,paclitaxel, pamidronate, pentostatin, plicamycin, procarbazine,steroids, streptozocin, STI-571, tamoxifen, temozolomide, teniposide,tetrazine, thioguanine, thiotepa, tomudex, topotecan, treosulphan,trimetrexate, vinblastine, vincristine, vindesine, vinorelbine, VP-16,xeloda, asparaginase, AIN-457, bapineuzumab, belimumab, brentuximab,briakinumab, canakinumab, cetuximab, dalotuzumab, denosumab,epratuzumab, estafenatox, farletuzumab, figitumumab, galiximab,gemtuzumab, girentuximab (WX-G250), herceptin, ibritumomab, inotuzumab,ipilimumab, mepolizumab, muromonab-CD3, naptumomab, necitumumab,nimotuzumab, ocrelizumab, ofatumumab, otelixizumab, ozogamicin,pagibaximab, panitumumab, pertuzumab, ramucirumab, reslizumab,rituximab, REGN88, solanezumab, tanezumab, teplizumab, tiuxetan,tositumomab, trastuzumab, tremelimumab, vedolizumab, zalutumumab,zanolimumab, 5FC, accutane hoffmann-la roche, AEE788 novartis, AMG-102,anti neoplaston, AQ4N (Banoxantrone), AVANDIA (Rosiglitazone Maleate),avastin (Bevacizumab) genetech, BCNU, biCNU carmustine, CCI-779, CCNU,CCNU lomustine, celecoxib (Systemic), chloroquine, cilengitide (EMD121974), CPT-11 (CAMPTOSAR, Irinotecan), dasatinib (BMS-354825,Sprycel), dendritic cell therapy, etoposide (Eposin, Etopophos,Vepesid), GDC-0449, gleevec (imatinib mesylate), gliadel wafer,hydroxychloroquine, IL-13, IMC-3G3, immune therapy, iressa (ZD-1839),lapatinib (GW572016), methotrexate for cancer (Systemic), novocure,OSI-774, PCV, RAD001 novartis (mTOR inhibitor), rapamycin (Rapamune,Sirolimus), RMP-7, RTA 744, simvastatin, sirolimus, sorafenib, SU-101,SU5416 sugen, sulfasalazine (Azulfidine), sutent (Pfizer), TARCEVA(erlotinib HCl), taxol, TEMODAR schering-plough, TGF-B anti-sense,thalomid (thalidomide), topotecan (Systemic), VEGF trap, VEGF-trap,vorinostat (SAHA), XL 765, XL184, XL765, zarnestra (tipifarnib), ZOCOR(simvastatin), cyclophosphamide (Cytoxan), (Alkeran), chlorambucil(Leukeran), thiopeta (Thioplex), busulfan (Myleran), procarbazine(Matulane), dacarbazine (DTIC), altretamine (Hexalen), clorambucil,cisplatin (Platinol), ifosafamide, methotrexate (MTX), 6-thiopurines(Mercaptopurine [6-MP], Thioguanine [6-TG]), mercaptopurine(Purinethol), fludarabine phosphate, (Leustatin), flurouracil (5-FU),cytarabine (ara-C), azacitidine, vinblastine (Velban), vincristine(Oncovin), podophyllotoxins (etoposide {VP-16} and teniposide {VM-26}),camptothecins (topotecan and irinotecan), taxanes such as paclitaxel(Taxol) and docetaxel (Taxotere), (Adriamycin, Rubex, Doxil),dactinomycin (Cosmegen), plicamycin (Mithramycin), mitomycin:(Mutamycin), bleomycin (Blenoxane), estrogen and androgen inhibitors(Tamoxifen), gonadotropin-releasing hormone agonists (Leuprolide andGoserelin (Zoladex)), aromatase inhibitors (Aminoglutethimide andAnastrozole (Arimidex)), amsacrine, asparaginase (El-spar), mitoxantrone(Novantrone), mitotane (Lysodren), retinoic acid derivatives, bonemarrow growth factors (sargramostim and filgrastim), amifostine,pemetrexed, decitabine, iniparib, olaparib, veliparib, everolimus,vorinostat, entinostat (SNDX-275), mocetinostat (MGCD0103), panobinostat(LBH589), romidepsin, valproic acid, flavopiridol, olomoucine,roscovitine, kenpaullone, AG-024322 (Pfizer), fascaplysin, ryuvidine,purvalanol A, NU2058, BML-259, SU 9516, PD-0332991, P276-00,geldanamycin, tanespimycin, alvespimycin, radicicol, deguelin, BIIB021,cis-imidazoline, benzodiazepinedione, spiro-oxindoles, isoquinolinone,thiophene, 5-deazaflavin, tryptamine, aminopyridine, diaminopyrimidine,pyridoisoquinoline, pyrrolopyrazole, indolocarbazole, pyrrolopyrimidine,dianilinopyrimidine, benzamide, phthalazinone, tricyclic indole,benzimidazole, indazole, pyrrolocarbazole, isoindolinone, morpholinylanthracycline, a maytansinoid, ducarmycin, auristatins, calicheamicins(DNA damaging agents), α-amanitin (RNA polymerase II inhibitor),centanamycin, pyrrolobenzodiazepine, streptonigtin, nitrogen mustards,nitrosorueas, alkane sulfonates, pyrimidine analogs, purine analogs,antimetabolites, folate analogs, anthracyclines, taxanes, vincaalkaloids, topoisomerase inhibitors, hormonal agents, and anycombination thereof.

Active agents useable in accordance with the present disclosure are notlimited to those drug classes or particular agents enumerated above.Different discovery platforms continue to yield new agents that aredirected at unique molecular signatures of cancer cells; indeed,thousands of such chemical and biological drugs have been discovered,only some of which are listed here. Yet, the surprising capability ofintact, bacterially derived minicells and killed bacterial cells toaccommodate packaging of a diverse variety of active agents, hydrophilicor hydrophobic, means that essentially any such drug, when packaged inminicells, has the potential to treat a cancer, pursuant to the findingsin the present disclosure.

Illustrative of the class of anti-neoplastic agents are radionuclides,chemotherapy drugs, and functional nucleic acids, including but notlimited to regulatory RNAs. Members of the class are discussed furtherbelow.

i. Radionuclides

A “radionuclide” is an atom with an unstable nucleus, i.e., onecharacterized by excess energy available to be imparted either to anewly created radiation particle within the nucleus or to an atomicelectron. Radionuclides herein may also be referred to as“radioisotopes,” “radioimaging agents,” or “radiolabels.” Radionuclidescan be used imaging and/or therapeutic purposes. They can be containedwithin the minicell or attached to a ligand, peptide, or glycolipid onthe minicell outer surface. Attachments may be directly or via a linker,a linker containing a chelating moiety comprising chelators such asmercaptoacetyltriglycine (MAG3), DOTA, EDTA, HYNIC, DTPA, or crownethers may be used. The chelators may be attached directly the minicellsurface component or attached to the minicell via a linker. Numerousradionuclides are known in the art, and a number of them are known to besuitable for medical use, such as yttrium-90, technetium-99m,iodine-123, iodine-124, iodine-125, iodine-131, rubidium-82,thallium-201, gallium-67, fluorine-18, xenon-133, and indium-111.

Thus, in some embodiments, the radioisotope comprises a radioisotopeselected from the group consisting of yttrium-90, yttrium-86,terbium-152, terbium-155, terbium-149, terbium-161, technetium-99m,iodine-123, iodine-131, rubidium-82, thallium-201, gallium-67,fluorine-18, copper-64, gallium-68, xenon-133, indium-111, lutetium-177,and any combination thereof.

Radioisotopes useful for attaching to minicells for both imaging andtherapeutic purposes include, for example, Iodine-131 and lutetium-177,which are gamma and beta emitters. Thus, these agents can be used forboth imaging and therapy.

Different isotopes of the same element, for example, iodine-123 (gammaemitter) and iodine-131 (gamma and beta emitters), can also be used forboth imaging and therapeutic purposes (Gerard and Cavalieri, 2002;Alzahrani et al., 2012).

Newer examples are yttrium-86/yttrium-90 or terbium isotopes (Tb): ¹⁵²Tb(beta plus emitter), ¹⁵⁵Tb (gamma emitter), ¹⁴⁹Tb (alpha emitter), and¹⁶¹Tb (beta minus particle) (Müller et al., 2012; Walrand et al., 2015).

Nuclear imaging utilizes gamma and positron emitters (β+). Gammaemitters, such as technetium-99m (^(99m)Tc) or iodine-123 (¹²³I), can belocated using gamma cameras (planar imaging) or SPECT (single photonemission computed tomography) (Holman and Tumeh, 1990).

The tissue penetration of these particles is proportional to the energyof the radioisotopes (Kramer-Marek and Capala, 2012). Beta particleshave a potential cytocidal effect, but they also spare the surroundinghealthy tissue due to having a tissue penetration of only a fewmillimeters. Commonly used beta emitters in routine nuclear oncologypractices include lutetium-177 (¹⁷⁷Lu, tissue penetration: 0.5-0.6 mm,maximum: 2 mm, 497 keV, half-life: 6.7 days) and yttrium-90 (⁹⁰Y, tissuepenetration: mean 2.5 mm, maximum: 11 mm, 935 keV, half-life: 64 hours)(Teunissen et al., 2005; Kwekkeboom et al., 2008; Ahmadzadehfar et al.,2010; Pillai et al., 2013; Ahmadzadehfar et al., 2016).

Radionuclides have found extensive use in nuclear medicine, particularlyas beta-ray emitters for damaging tumor cells. In some embodiments,radionuclides are suitably employed as the anti-neoplastic agents.

Radionuclides can be associated with intact, bacterially derivedminicells by any known technique. Thus, a protein or otherminicell-surface moiety (see below) can be labeled with a radionuclide,using a commercially available labeling means, such as use of PierceIodination reagent, a product of Pierce Biotechnology Inc. (Rockford,Ill.), detailed in Rice et al., Semin. Nucl. Med., 41, 265-282 (2011).Alternatively, radionuclides can be incorporated into proteins that areinside minicells.

In the latter situation, a minicell-producing bacterial strain istransformed with plasmid DNA encoding foreign protein. When minicellsare formed during asymmetric cell division, several copies of theplasmid DNA segregate into the minicell cytoplasm. The resultantrecombinant minicells are incubated in the presence of radiolabeledamino acids under conditions such that foreign protein expressed insidethe minicell, from the plasmid DNA, incorporates into theradionuclide-carrying amino acids. Pursuant to the protocol ofClark-Curtiss and Curtiss, Methods Enzymol., 101: 347-362 (1983), forinstance, recombinant minicells are incubated in minimal growth mediumthat contains ^(35S)methionine, whereby newly expressed, plasmid-encodedproteins incorporate the ^(35S)methionine. A similar approach can beused so that recombinant minicells become packaged with otherradiolabels, as desired.

Oligosaccharides on the minicell surface also can be radiolabeled using,for example, well-established protocols described by Fukuda, Curr.Protocols Molec. Biol. (Suppl. 26), 17.5.1-17.5.8 (1994). Illustrativeof such oligosaccharides that are endemic to minicells is theO-polysaccharide component of the lipopolysaccharide (LPS) found on thesurface of minicells derived from Gram-negative bacteria (see below).

A preferred methodology in this regard is to radiolabel a bispecificantibody used as a tumor targeting ligand that is used to targetminicells to specific tumors. See US Patent Publication 2007/0237744,the contents of which are incorporated herein by reference. That is, thebispecific antibody “coated” on a minicell exposes a significant amountof additional surface protein for radiolabeling. Accordingly, it ispossible to achieve a higher specific activity of the radiolabelassociated with the antibody-coated minicell. By contrast, theradiolabeling of non-coated minicells, i.e., when the radionuclidelabels only endemic moieties, can result in weaker labeling (lowerspecific activity). In one embodiment, this weaker labeling is thoughtto occur because the outer membrane-associated proteins of minicellsderived from Gram-negative bacteria are masked by LPS, which, as furtherdiscussed below, comprises long chains of O-polysaccharide covering theminicell surface.

For treating a tumor, a composition of the disclosure would be deliveredin a dose or in multiple doses that affords a level of in-tumorirradiation that is sufficient at least to reduce tumor mass, if noteliminate the tumor altogether. The progress of treatment can bemonitored along this line, on a case-by-case basis. In general terms,however, the amount of radioactivity packaged in the compositiontypically will be on the order of about 30 to about 50 Gy, although theinvention also contemplates a higher amount of radioactivity, such asfor example about 50 to about 200 Gy, which gives an overall rangebetween about 30 Gy and about 200 Gy.

In some instances, the amount of radioactivity packaged in thecomposition can be even lower than mentioned above, given the highlyefficient and specific delivery of the minicell-bourne radionuclides toa tumor. Accordingly, in one aspect the composition comprises from about20 to about 40 Gy, or about 10 to about 30 Gy, or about 1 to about 20Gy, or less than about 10 Gy.

Some tumor targeting ligands may include a radioisotope that functionsto deliver radiation to the tumor while the ligand binds the tumor cell.In some embodiments, the ligand comprises Arg-Gly-Asp (RGD) peptide,bombesin (BBN)/gastrin-releasing peptide (GRP), cholecystokinin(CCK)/gastrin peptide, α-melanocyte-stimulating hormone (α-MSH),neuropeptide Y (NPY), neutrotensin (NT), [⁶⁸Ga]Ga-PSMA-HBED-CC([⁶⁸Ga]Ga-PSMA-11 [PET]), [¹⁷⁷Lu]Lu/[⁹⁰Y]Y-J591, [1²³I]I-MIP-1072,[131I]I-MIP-1095, ⁶⁸Ga or ¹⁷⁷Lu labeled PSMA-I&T, ⁶⁸Ga or ¹⁷⁷Lu labeledDKFZ-PSMA-617 (PSMA-617), somatostatin (SST) peptide, substance P, T140,tumor molecular targeted peptide 1 (TMTP1), vasoactive intestinalpeptide (VIP), or any combination thereof.

In some embodiments, the radioisotope is conjugated to the tumortargeting ligand. In some embodiments, the conjugation is via a linker.In some embodiments, the tumor targeting ligand comprises a peptidecomprising functional group(s) for conjugation of a radioisotope orchelator moiety that chelates a radioisotope. The functional groups ofpeptides available for conjugation include but are not limited to theε-amino group on lysine side chains, the guanidinium group on arginineside chains, the carboxyl groups on aspartic acid or glutamic acid, thecysteine thiol, and the phenol on tyrosine. The most common conjugationreactions are carbodiimide/N-hydroxysuccinimidyl (EDC/NHS) mediatedcarboxyl and amine coupling, maleimide conjugation to thiol groups, anddiazonium modification of the phenol on tyrosine. The representativechemistries to couple peptides with imaging moieties can be found in anumber of reviews (Erathodiyil and Ying, 2011; Takahashi et al., 2008).

In some embodiments, the radioisotope functions as a radioimaging agent.Several radioisotopes have been used for peptide labeling including⁹⁹mTc, ¹²³I, and ¹¹¹In for SPECT imaging and ¹⁸F, ⁶⁴Cu and ⁶⁸Ga for PETimaging (Chatalic et al., 2015). Generally, these radioisotopes areattached to the peptides via chelators. Some widely-used chelators aredescribed in (Sun et al., 2017). Most therapeutic radiopharmaceuticalsare labeled with beta-emitting isotopes (β-).

The minicells of the present invention, targeted to the tumor cells willalso deliver targeted radiation from the radioisotope to the tumor cellto which the minicell is bound. In some embodiments, the radioisotopefunctions as a therapeutic radiation emitting agent, and wherein theamount of radiation provided by the radioisotope is sufficient toprovide a therapeutic effect on the tumor. In some embodiments, thetherapeutic effect is a reduction in tumor size. The tumor may bereduced in size by about 100%, about 90%, about 80%, about 70%, about60%, about 50%, about 40%, about 30%, about 20%, about 10%, or about 5%.

Radiolabeled phosphonates have a high bone affinity and can be used forimaging and palliation of painful bone metastases. Depending on thedegree of osseous metabolism, the tracer accumulates via adhesion tobones and, preferably, to osteoblastic bone metastases. Therapy planningrequires a bone scintigraphy with technetium-99m-hydroxyethylidenediphosphonate (HEDP) to estimate the metabolism and the extent of themetastases involvement. Bisphosphonate HEDP can be labeled for therapyeither with rhenium-186 (beta-emitter, half-life: 89 hours, 1.1 MeVmaximal energy, maximal range: 4.6 mm) or rhenium-188 (beta-emitter [to85%, 2.1 MeV] and gamma-emitter [to 15%, 155 keV], half-life: 16.8hours, maximal range in soft tissue: 10 mm) (Palmedo, 2007). Newpromising radiopharmaceuticals for bone palliation therapy includeradiolabeled complexes of zoledronic acid. Zoledronic acid belongs to anew, most potent generation of bisphosphonates with cyclic side chains.The bone affinity of zoledronic acid labeled with scandium-46 orlutetium-177 has shown excellent absorption (98% for[177Lu]Lu-zoledronate and 82% for [46Sc]Sc-zoledronate), which is muchhigher than of bisphosphonates labeled with samarium-153 (maximum: 67%)(Majkowska et al., 2009). These bisphosphonates can be conjugated tointact minicells for use as diagnostics or treatment for bonemetastasis.

ii. Chemotherapy Drugs

An antineoplastic agent employed in the present disclosure can also be achemotherapy drug. In this description, “chemotherapeutic drug,”“chemotherapeutic agent,” and “chemotherapy” are employedinterchangeably to connote a drug that has the ability to kill ordisrupt a neoplastic cell. A chemotherapeutic agent can be a smallmolecule drug or a biologic drug, as further detailed below.

The “small molecule drug” subcategory encompasses compoundscharacterized by having (i) an effect on a biological process and (ii) alow molecular weight as compared to a protein or polymericmacromolecule. Small molecule drugs typically are about 800 Daltons orless, with a lower limit of about 150 Daltons, as illustrated byTemodar® (temozolomide), at about 194 Daltons, which is used to treatglioblastoma and other types of brain cancer. In this context “about”indicates that the qualified molecular-weight value is subject tovariances in measurement precision and to experimental error on theorder of several Daltons or tens of Daltons. Thus, a small molecule drugcan have a molecular weight of about 900 Daltons or less, about 800 orless, about 700 or less, about 600 or less, about 500 or less, or about400 Daltons or less, e.g., in the range of about 150 to about 400Daltons. More specifically, a small molecule drug can have a molecularweight of about 400 Daltons or more, about 450 Daltons or more, about500 Daltons or more, about 550 Daltons or more, about 600 Daltons ormore, about 650 Daltons or more, about 700 Daltons or more, or about 750Daltons or more. In another embodiment, the small molecule drug packagedinto the minicells has a molecular weight between about 400 and about900 Daltons, between about 450 and about 900 Daltons, between about 450and about 850 Daltons, between about 450 and about 800 Daltons, betweenabout 500 and about 800 Daltons, or between about 550 and about 750Daltons.

Specifically, suitable small molecule drugs include but are not limitedto those listed above, such as nitrogen mustards, nitrosorueas,ethyleneimine, alkane sulfonates, tetrazine, platinum compounds,pyrimidine analogs, purine analogs, anti-metabolites, folate analogs,anthracyclines, taxanes, vinca alkaloids, and topoisomerase inhibitors,inter alia. Accordingly, a small molecule drug for use in the presentinvention can be selected from among any of the following, inter alia:enediynes, such as dynemicin A, unicalamycin, calicheamicin yl andcalicheamicin-theta-1; meayamicin, a synthetic analog of FR901464;benzosuberene derivatives as described, for example, by Tanpure et al.,Bioorg. Med. Chem., 21: 8019-32 (2013); auristatins, such as auristatinE, mono-methyl auristatin E (MMAE), and auristatin F, which aresynthetic analogs of dolastatin; duocarmysins such as duocarmycin SA andCC-1065; maytansine and its derivatives (maytansinoids), such as DM1 andDM4; irinotecan (Camptosar®) and other topoisomerase inhibitors, such astopotecan, etoposide, mitoxantrone and teniposide; and yatakemycin, thesynthesis of which is detailed by Okano et at, 2006.

More particularly, any one or more or all of the specific small moleculedrugs detailed herein are illustrative of those suitable for use in thisinvention: actinomycin-D, alkeran, ara-C, anastrozole, BiCNU,bicalutamide, bisantrene, bleomycin, busulfan, capecitabine (Xeloda®),carboplatin, carboplatinum, carmustine, CCNU, chlorambucil, cisplatin,cladribine, CPT-11, cyclophosphamide, cytarabine, cytosine arabinoside,cytoxan, dacarbazine, dactinomycin, daunorubicin, dexrazoxane,docetaxel, doxorubicin, DTIC, epirubicin, ethyleneimine, etoposide,floxuridine, fludarabine, fluorouracil, flutamide, fotemustine,gemcitabine, hexamethylamine, hydroxyurea, idarubicin, ifosfamide,irinotecan, lomustine, mechlorethamine, melphalan, mercaptopurine,methotrexate, mitomycin, mitotane, mitoxantrone, oxaliplatin,paclitaxel, pamidronate, pentostatin, plicamycin, procarbazine,streptozocin, STI-571, tamoxifen, temozolomide, teniposide, tetrazine,thioguanine, thiotepa, tomudex, topotecan, treosulphan, trimetrexate,vinblastine, vincristine, vindesine, vinorelbine, and VP-16.

For purposes of this description a “biologic drug” is defined, bycontrast, to denote any biologically active macromolecule that can becreated by a biological process, exclusive of “functional nucleicacids,” discussed below, and polypeptides that by size qualify as smallmolecule drugs, as defined above. The “biologic drug” subcategory thusis exclusive of and does not overlap with the small molecule drug andfunctional nucleic acid subcategories. Illustrative of biologic drugsare therapeutic proteins and antibodies, whether natural or recombinantor synthetically made, e.g., using the tools of medicinal chemistry anddrug design.

iii. Supertoxic Chemotherapy Drugs

Certain molecules that are designed for chemotherapeutic purposes failduring pre-clinical or clinical trials due to unacceptable toxicity. Thepresent inventors have shown that packaging a highly toxic or“supertoxic” chemotherapy drug in a minicell, followed by systemicdelivery to a tumor patient, results in delivery of the drug to tumorcells. Further, even after the tumor cells are broken up and thedrug-containing cytoplasm is released to the nearby normal tissue, theresult is not toxicity to normal tissue. This is because the drug isalready bound to the tumor cellular structures, such as DNA, and can nolonger attack normal cells. Accordingly, the present invention isparticularly useful for delivery of highly toxic (“supertoxic”)chemotherapy drugs to a cancer patient.

When cancer subjects have exhausted all treatment options, the tumorsare likely to have reached a stage of considerable heterogeneity with ahigh degree of resistance to conventional cytotoxic drugs. “Highly toxicchemotherapy drug” or “supertoxic chemotherapy drugs” in thisdescription refer to chemotherapy drugs that can overcome the resistanceto conventional drugs due to their relatively low lethal dose to normalcells as compared to their effective dose for cancer cells.

Thus, in one aspect a highly toxic chemotherapy drug has a median lethaldose (LD₅₀) that is lower than its median effective dose (ED₅₀) for atargeted cancer. For instance, a highly toxic or supertoxic chemotherapydrug can have an LD₅₀ that is lower than about 500%, 400%, 300%, 250%,200%, 150%, 120%, or 100% of the ED₅₀ of the drug for a targeted cancer.In another aspect, a highly toxic or supertoxic chemotherapy drug has amaximum sub-lethal dose (i.e., the highest dose that does not causeserious or irreversible toxicity) that is lower than its minimumeffective dose, e.g., about 500%, about 400%, about 300%, about 250%,about 200%, about 150%, about 120%, about 100%, about 90%, about 80%,about 70%, about 60% or about 50% of the minimum effective dose. In oneembodiment, the targeted cancer can be, for example, (1) a cancer typefor which the drug is designed, (2) the first cancer type in which apre-clinical or clinical trial is run for that drug, or (3) a cancertype in which the drug shows the highest efficacy among all testedcancers.

Illustrative, non-limiting examples of supertoxic chemotherapy drugsinclude but are not limited to maytansinoids, duocarmycins, morpholinylanthracycline, and their derivatives. Maytansinoids (molecular weight:about 738 Daltons) are a group of chemical derivatives of maytansine,having potent cytotoxicity. Although considered unsafe for human patientuse, due to toxicity concerns, maytansinoids are suitable for deliveryto tumor patients via minicells, pursuant to the present invention.Duocarmycins (molecular weight: about 588 Daltons) are a series ofrelated natural products, first isolated from Streptomyces bacteria.They also have potent cytotoxicity but are considered as unsafe forhuman use. Like maytansinoids, duocarmycins are suitable chemotherapydrugs for use in the invention.

Illustrative as well are compounds in the class of morpholinylanthracycline derivatives described in international patent applicationWO 1998/002446. Among such derivatives are nemorubicin(3′-deamino-3′-[2(S)-methoxy-4-morpholinyl]doxorubicin) (MMDX), and itsmajor metabolite PNU-159682(3′-deamino-3″-4′-anhydro-[2″(S)-methoxy-3″(R)-hydroxy-4″-morpholinyl-]doxorubicin),as well as four other such derivatives described in U.S. Pat. No.8,470,984, the contents of which are incorporated here by reference:3′-deamino-3″-4′-anhydro-[2″(S)-methoxy-3″(R)-hydroxy-4″-morpholinyl]-idarubicin;3′-deamino-3″-4′-anhydro-[2″(S)-methoxy-3′(R)-hydroxy-4′-morpholinyl]-daunorubicin;3′-deamino-3″-4′-anhydro-[2″(S)-methoxy-3′(R)-hydroxy-4′-morpholinyl]-caminomycin;and3′-deamino-3″-4′-anhydro-[2″(S)-ethoxy-3(R)-hydroxy-4″-morpholinyl]d-oxorubicin.

In an exemplary embodiment of the present disclosure, the minicellcomprises the supertoxic chemotherapy drug3′-deamino-3″,4′-anhydro-[2″(S)-methoxy-3′(R)-oxy-4-morpholinyl]doxorubicin(PNU-159682). The present inventors discovered that PNU-159682 is apotent drug that appears to overcome drug resistance in a number ofdifferent tumor cell lines and is much more potent than a range ofconventional chemotherapeutics in cytotoxicity assays against manydifferent tumor cell lines. See Examples 8 and 9. Further, it was shownin in vivo mouse xenograft experiments that human tumor xenograftsresistant to doxorubicin can be treated effectively with IVadministration of EGFR-targeted and PNU-159682-loaded EDVs. See Example11. Remarkably, PNU-159682-loaded EDVs combined with type I interferonagonists was found to be well-tolerated and to provide synergistic andimproved anti-cancer effect in a late-stage pancreatic cancer patient.See Example 12. Accordingly, in one embodiment of the present inventiona composition comprises an EGFR-targeted minicell comprising PNU-159682as an active anticancer drug.

Other suitable cancer chemotherapy drugs that may exhibit supertoxicchemotherapy properties include auristatins, calicheamicins (DNAdamaging agents), α-amanitin (RNA polymerase II inhibitor),centanamycin, geldanamycin, pyrrolobenzodiazepine, streptonigtin,nitrogen mustards, nitrosorueas, ethyleneimine, alkane sulfonates,tetrazine, platinum compounds, pyrimidine analogs, purine analogs,antimetabolites, folate analogs, anthracyclines, taxanes, vincaalkaloids, topoisomerase inhibitors, and hormonal agents, inter alia.

iv. Biologic Chemotherapy Drugs

In another aspect, the minicells may comprise a biologic chemotherapydrug. Examples of such drugs include but are not limited toasparaginase, AIN-457, bapineuzumab, belimumab, brentuximab,briakinumab, canakinumab, cetuximab, dalotuzumab, denosumab,epratuzumab, estafenatox, farletuzumab, figitumumab, galiximab,gemtuzumab, girentuximab (WX-G250), ibritumomab, inotuzumab, ipilimumab,mepolizumab, muromonab-CD3, naptumomab, necitumumab, nimotuzumab,ocrelizumab, ofatumumab, otelixizumab, ozogamicin, pagibaximab,panitumumab, pertuzumab, ramucirumab, reslizumab, rituximab, REGN88,solanezumab, tanezumab, teplizumab, tiuxetan, tositumomab, trastuzumab(Herceptin®), tremelimumab, vedolizumab, zalutumumab, and zanolimumab.

v. Functional Nucleic Acids

“Functional nucleic acid” refers to a nucleic acid molecule that, uponintroduction into a host cell, specifically interferes with expressionof a protein. With respect to treating cancer, in accordance with thedisclosure, it is preferable that a functional nucleic acid payloaddelivered to cancer cells via intact, bacterially derived minicellsinhibits a gene that promotes tumor cell proliferation, angiogenesis orresistance to chemotherapy and/or that inhibits apoptosis or cell-cyclearrest; i.e., a “cancer-promoting gene.”

In general, functional nucleic acid molecules used in this disclosurehave the capacity to reduce expression of a protein by interacting witha transcript for a protein. This category of minicell payload for thedisclosure includes regulatory RNAs, such as siRNA, shRNA, short RNAs(typically less than 400 bases in length), micro-RNAs (miRNAs),ribozymes and decoy RNA, antisense nucleic acids, and LincRNA, interalia. In this regard, “ribozyme” refers to an RNA molecule having anenzymatic activity that can repeatedly cleave other RNA molecules in anucleotide base sequence-specific manner. “Antisense oligonucleotide”denotes a nucleic acid molecule that is complementary to a portion of aparticular gene transcript, such that the molecule can hybridize to thetranscript and block its translation. An antisense oligonucleotide cancomprise RNA or DNA. The “LincRNA” or “long intergenic non-coding RNA”rubric encompasses non-protein coding transcripts longer than 200nucleotides. LincRNAs can regulate the transcription, splicing, and/ortranslation of genes, as discussed by Khalil et al., 2009.

Each of the types of regulatory RNA can be the source of functionalnucleic acid molecule that inhibits a tumor-promoting gene as describedabove and, hence, that is suitable for use according to the presentdisclosure. Thus, in one embodiment of the disclosure the intactminicells carry siRNA molecules mediating a post-transcriptional,gene-silencing RNA interference (RNAi) mechanism, which can be exploitedto target tumor-promoting genes. For example, see MacDiarmid et al.,2009 (antibody-presenting minicells deliver, with chemotherapy drug,siRNAs that counter developing resistance to drug), and Oh and Park,Advanced Drug Delivery Rev., 61: 850-62 (2009) (delivery of therapeuticsiRNAs to treat breast, ovarian, cervical, liver, lung and prostatecancer, respectively).

As noted, “siRNA” generally refers to double-stranded RNA molecules fromabout 10 to about 30 nucleotides long that are named for their abilityspecifically to interfere with protein expression. Preferably, siRNAmolecules are about 12 to about 28 nucleotides long, more preferablyabout 15 to about 25 nucleotides long, still more preferably about 19 toabout 23 nucleotides long and most preferably about 21 to about 23nucleotides long. Therefore, siRNA molecules can be, for example, about12, about 13, about 14, about 15, about 16, about 17, about 18, about19, about 20, about 21, about 22, about 23, about 24, about 25, about26, about 27, about 28, or about 29 nucleotides in length.

The length of one strand designates the length of an siRNA molecule. Forinstance, an siRNA that is described as 21 ribonucleotides long (a21-mer) could comprise two opposing strands of RNA that anneal for 19contiguous base pairings. The two remaining ribonucleotides on eachstrand would form an “overhang.” When a siRNA contains two strands ofdifferent lengths, the longer of the strands designates the length ofthe siRNA. For instance, a dsRNA containing one strand that is 21nucleotides long and a second strand that is 20 nucleotides long,constitutes a 21-mer.

Tools to assist the design of siRNA specifically and regulatory RNAgenerally are readily available. For instance, a computer-based siRNAdesign tool is available on the internet at www.dharmacon.com.

In another preferred embodiment, the intact minicells of the presentdisclosure carry miRNAs, which, like siRNA, are capable of mediating apost-transcriptional, gene-silencing RNA interference (RNAi) mechanism.Also like siRNA, the gene-silencing effect mediated by miRNA can beexploited to target tumor-promoting genes. For example, see Kota et al.,2009 (delivery of a miRNA via transfection resulted in inhibition ofcancer cell proliferation, tumor-specific apoptosis and dramaticprotection from disease progression without toxicity in murine livercancer model), and Takeshita et al., 2010 (delivery of synthetic miRNAvia transient transfection inhibited growth of metastatic prostate tumorcells on bone tissues).

Although both mediate RNA interference, miRNA and siRNA have noteddifferences. In this regard, “miRNA” generally refers to a class ofabout 17 to about 27-nucleotide single-stranded RNA molecules (insteadof double-stranded as in the case of siRNA). Therefore, miRNA moleculescan be, for example, about 17, about 18, about 19, about 20, about 21,about 22, about 23, about 24, about 25, about 26, or about 27nucleotides in length. Preferably, miRNA molecules are about 21 to about25 nucleotide long.

Another difference between miRNAs and siRNAs is that the formergenerally do not fully complement the mRNA target. In contrast, siRNAmust be completely complementary to the mRNA target. Consequently, siRNAgenerally results in silencing of a single, specific target, while miRNAis promiscuous.

Additionally, although both are assembled into RISC (RNA-inducedsilencing complex), siRNA and miRNA differ in their respective initialprocessing before RISC assembly. These differences are described indetail in Chu et al., 2006; and Gregory et al., 2006. A number ofdatabases serve as miRNA depositories. For example, see miRBase(www.mirbase.org) and tarbase(http://diana.cslab.ece.ntua.gr/DianaToolsNew/index.php?r=tarbase/index).In conventional usage, miRNAs typically are named with the prefix“-mir,” combined with a sequential number. For instance, a new miRNAdiscovered after mouse mir-352 will be named mouse “mir-353.” Again,tools to assist the design of regulatory RNA including miRNA are readilyavailable. In this regard, a computer-based miRNA design tool isavailable on the internet at wmd2.weigelworld.org/cgi-bin/mirnatools.pl.

It is a discovery of the present inventors that miRNA16a can beadministered by targeted minicell-mediated delivery to mesothelioma andAdreno-Cortical cancer cells. See Example 7. Once internalized by thecancer cells, the miRNA16a was found to potently inhibit cancer cellproliferation. Accordingly, in some embodiments the minicells of thepresent disclosure comprise miRNA16a. Other microRNAs useful ininhibiting the proliferation of neoplastic cells include mir-34 familyand let-7 family.

As noted above, a functional nucleic acid employed in the compositionsof the invention can inhibit a gene that promotes tumor cellproliferation, angiogenesis or resistance to chemotherapy. The inhibitedgene also can itself inhibit apoptosis or cell cycle arrest. Examples ofgenes that can be targeted by a functional nucleic acid are providedbelow.

Functional nucleic acids of the disclosure preferably target the gene ortranscript of a protein that promotes drug resistance, inhibitsapoptosis or promotes a neoplastic phenotype. Successful application offunctional nucleic acid strategies in these contexts have been achievedin the art, but without the benefits of minicell vectors. See, e.g.,Sioud, Trends Pharmacol. Sci., 2004; Caplen, Expert Opin. Biol. Ther.,2003; Nieth et al., 2003; Caplen and Mousses, 2003; Duxbury et al.,2004; Yague et aL, 2004; and Duan et aL, 2004.

Proteins that contribute to drug resistance constitute preferred targetsof functional nucleic acids. The proteins may contribute to acquireddrug resistance or intrinsic drug resistance. When diseased cells, suchas tumor cells, initially respond to drugs, but become refractory onsubsequent treatment cycles, the resistant phenotype is acquired. Usefultargets involved in acquired drug resistance include ATP bindingcassette transporters such as P-glycoprotein (P-gp, P-170, PGY1, MDR1,ABCB 1, MDR-associated protein, Multidrug resistance protein 1), MDR-2and MDR-3. MRP2 (multi-drug resistance associated protein), BCR-ABL(breakpoint cluster region—Abelson protooncogene), a STI-571resistance-associated protein, lung resistance-related protein,cyclooxygenase-2, nuclear factor kappa, XRCC1 (X-ray cross-complementinggroup 1), ERCC1 (excision cross-complementing gene), GSTP1 (glutathioneS-transferase), mutant .beta.-tubulin, and growth factors such as IL-6are additional targets involved in acquired drug resistance.

Particularly useful targets that contribute to drug resistance includeATP binding cassette transporters such as P-glycoprotein, MDR-2, MDR-3,BCRP, APT11a, and LRP. Useful targets also include proteins that promoteapoptosis resistance. These include Bcl-2 (B cell leukemia/lymphoma),Bcl-X_(L), A1/Bfl 1, focal adhesion kinase, dihydrodiol dehydrogenase,and p53 mutant protein.

Useful targets further include oncogenic and mutant tumor suppressorproteins. Illustrative of these are .beta.-Catenin, PKC-.alpha. (proteinkinase C), C-RAF, K-Ras (V12), DP97 Dead box RNA helicase, DNMT1 (DNAmethyltransferase 1), FLIP (Flice-like inhibitory protein), C-Sfc,53BPI, Polycomb group protein EZH2 (Enhancer of zeste homologue), ErbB1,HPV-16 E5 and E7 (human papillomavirus early 5 and early 7), Fortilin &MCI1P (Myeloid cell leukemia 1 protein), DIP13.alpha. (DDC interactingprotein 13a), MBD2 (Methyl CpG binding domain), p21, KLF4 (Kruppel-likefactor 4), tpt/TCTP (Translational controlled tumor protein), SPK1 andSPK2 (Sphingosine kinase), P300, PLK1 (Polo-like kinase-1), Trp53, Ras,ErbB1, VEGF (Vascular endothelial growth factor), BAG-1 (BCL2-associatedathanogene 1), MRP2, BCR-ABL, STI-571 resistance-associated protein,lung resistance-related protein, cyclooxygenase-2, nuclear factor kappa,XRCC1, ERCC1, GSTP1, mutant—β-tubulin, and growth factors.

Also useful as targets are global regulatory elements exemplified by thecytoplasmic polyadenylation element binding proteins (CEPBs). Forinstance, CEPB4 is overexpressed in glioblastoma and pancreatic cancers,where the protein activates hundreds of genes associated with tumorgrowth, and it is not detected in healthy cells (Oritz-Zapater et aL,2011). In accordance with the present description, therefore, treatmentof a glioblastoma could be effected via administration of a compositioncontaining intact, bacterially derived minicells that encompass an agentthat counters overexpression of CEPB4, such as an siRNA or otherfunctional nucleic acid molecule that disrupts CEPB4 expression by thetumor cells.

A further example of useful targets for functional nucleic acids includereplication protein A (RPA), a trimeric complex composed of 70-kDa(RPA1), 32-kDa (RPA2), and 14-kDa (RPA3) subunits, which is essentialfor DNA replication in all organisms. Iftode et al., 1999.

Other useful targets are those important for mitosis and for themaintenance of genomic stability. Examples included the Polo-like kinase(PLK1), which was found to be overexpressed in a broad range of cancercells. See Example 3, FIG. 12. The inventors of the present disclosurealso found that siRNA inhibiting Plk1 (siPlk1) expression inhibitsproliferation of mesothelioma and Adreno-Cortical cancer cells. SeeExample 10. Accordingly, in some embodiments, the minicells of thepresent disclosure comprise Plk1.

Other useful targets are those that are involved in DNA replication andrepair. Examples include ribonucleotide reductase (RR), which is apotential therapeutic target for cancer because it catalyzes theconversion of ribonucleoside 5′-diphosphates into their corresponding2′-deoxyribonucleoside 5′-triphosphates that are necessary for DNAreplication and repair. See D'Angiolella et al., 2012. Human RRcomprises two subunits, RRM1 and RRM2, and functional nucleic acids thattarget both subunits are useful in the present invention. The inventorsof the present disclosure showed that siRNA targeting RRM1 (siRRM1)potently inhibited mesothelioma and Adreno-Cortical cancer cellproliferation upon delivery with minicells. See Example 10. Accordingly,in some embodiments the minicell comprises siRNA, which inhibitsribonucleotide reductase M1 (RRM1) expression.

B. Type I Interferon Agonists

The present compositions can comprise a type 1 interferon agonist, i.e.,an agent that increases the level (e.g., the activity or expressionlevel) of type 1 interferons. Human type I interferons (IFNs) are alarge subgroup of interferon proteins that help regulate the activity ofthe immune system. Interferons bind to interferon receptors. All type IIFNs bind to a specific cell surface receptor complex known as the IFN-αreceptor (IFNAR), which consists of IFNAR1 and IFNAR2 chains. Mammaliantype I IFNs are designated IFN-α (alpha), IFN-β (beta), IFN-κ (kappa),IFN-δ(delta), IFN-ε (epsilon), IFN-τ (tau), IFN-ω (omega), and IFN-ξ,(zeta, also known as limitin).

i. Oligonucleotides

FIG. 2 shows a graphical depiction of an exemplary embodiment of aminicell comprising immunomodulatory, 60mer double-stranded DNA. Thepresent inventors discovered that delivery of a type I interferonagonist, such as double-stranded DNA with EGFR-targeted minicells, actsas an adjuvant (i.e., it enhances) anti-tumor efficacy of minicellsloaded with cytotoxic drugs. See Example 11. Thus, combining minicellspackaged with the supertoxic drug PNU-159682 resulted in enhancedanti-tumor effects, and this treatment was well-tolerated by alate-stage pancreatic cancer patient. See Example 12.

Expression of type I interferons (IFN) can be induced by deliveringdouble-stranded DNA to target cells. Specifically, innate immuneactivation by cytosolic DNA from microbial pathogens is a potent triggerof type I IFNs and pro-inflammatory cytokines mediated by cytosolic DNAsensors such as cGAMP, cyclic GMP-AMP synthetase (cGAS) and IFN gammainducible factor 16 (IFI16). See, e.g., Hansen et al., 2014; andUnterholzner et al., 2013. Post-binding to double stranded DNA, cGAS hasthe enzymatic capacity to produce the second messenger cyclic GMP-AMPwhich docks onto the endoplasmic reticulum-bound protein stimulator ofIFN genes (STING). Barber et al., 2011. This induces conformationalchanges that allow STING to homodimerize, migrate from the ER (Dobbs etal., 2015), and to recruit TANK-binding kinase 1 that phosphorylatesSTING, resulting in the transcription factor IFN regulatory factor 3that initiates expression of IFN. See Dobbs et al., 2015; Wang et al.,2014; and Liu et al., 2015. Thus, expression of type I IFNs can beinduced by delivering double-stranded DNA to target cells that can berecognized by cytosolic DNA sensors, as described above and in the citedreferences.

In some embodiments, the compositions disclosed herein include an intactminicell comprising an type I IFN agonist. In some embodiments, the typeI IFN agonist is an oligonucleotide suitable for DNA sensor mediatedinduction of type I IFN, as described herein. In some embodiments theoligonucleotide comprises a sequence of at least about 10, at leastabout 20, at least about 30, at least about 40, at least about 50, atleast about 60, at least about 70, at least about 80, at least about 90,at least about 100, at least about 110, at least about 120, at leastabout 130, at least about 140, at least about 150, at least about 160,at least about 170, at least about 180, at least about 190, or at leastabout 200 nucleotides. In another embodiment, the oligonucleotidecomprises a sequence of from about 10 up to about 200 nucleotides, orany amount in-between these two values. In some embodiments, theoligonucleotide comprises a sequence of at least about 40 nucleotides,at least about 50 nucleotides, or at least about 60 nucleotides.

In other embodiments, polynucleotide products of the enzymepolynucleotide phosphorylase (PNPase 1) may be used as syntheticinducers of IFN activity. Field et al., 1967. Similarly, the dsRNAmimetic polyinosinic:polycytidylic acid (poly(I:C)), was shown tofunction as an agonist for both TLR3 and MDA5. Alexopoulou et al., 2001;and Gitlin et al., 2006. Accordingly, in some embodiments, theoligonucleotide is a polynucleotide product of PNPase1, poly(I:C),poly-ICLC, imiquimod, imidazoquioline resquimod, orCpG-oligodeoxynucleotides.

Synthetic oligonucleotides can also be designed and used as agonists ofnucleic acid sensors. For example, TLR9-stimulatory synthetic CpGoligodeoxynucleotides (CpG-ODNs) were designed based on theimmune-stimulatory properties of bacterial DNA that, in contrast tohuman DNA, is rich in unmethylated CpG motifs. Krieg et al., 1995.Optimization of sequence features and backbone modifications led toCpG-ODN subtypes that preferentially activate either B cells or pDCs.Accordingly, it is contemplated herein that the CpG-ODN can bemethylated or unmethylated, or a combination of both.

There are a number of molecules that are known to be stimulators of typeI IFN secretion and these molecules along with their agonists aresuitable for delivery via minicells to elicit type I IFN secretion.These molecules include but are not limited to, double stranded RNA(dsRNA), poly(dA:dT) DNAs, double stranded Z-DNA and B-DNA, DNAs(dsDNAs) longer than 36 bp and DNA-RNA hybrids, bacterial secondmessenger cyclic-di-GMP, TLR3, TLR4, TLR7, TLR8 and TLR9 agonists, andSTING Agonists, which are more fully described below.

ii. Double-Stranded RNA (dsRNA)

Double-stranded RNA is an inducer of type I IFN. The RNA helicasesretinoic acid-inducible gene I (RIG-I) and melanomadifferentiation-associated gene 5 (MDA5) are cytoplasmic receptors thattrigger type I IFN secretion. These receptors (RIG-I-like receptors)transmit signals through the mitochondria-localized adaptor moleculeIPS-1 or MAVS and the kinases TBK1 and IKKi to activate IRF3 and inducetranscription of the type I IFN genes (Kawai and Akira, 2010). RIG-I andMDA5 respond to viral RNAs tri-phosphorylated in their 5′ ends (Leungand Amarasinghe, 2016; Lu et al., 2010; Marq et at, 2011; Wang et al.,2010).

iii. Poly(dA:dT) DNAs

RNA polymerase III is a cytosolic DNA sensor for poly(dA:dT) DNAs(Ablasser et al., 2009). In the cytosol, RNA polymerase III convertspoly(dA:dT) to RNA with 5′ tri-phosphorylation. The converted 5′-ppp RNAthen initiates the RIG-I-MAVS pathway and NFκB activation to elicit typeI IFN secretion.

iv. Double-Stranded Z-DNA and B-DNA

A cytosolic DNA sensor, DNA-dependent activator of IRFs (DAI) or Z-DNAbinding protein 1, is known to induce type I IFN in response to theright-handed dsDNA conformation (B-DNA) in a TBK1- and IRF3-mediatedmechanism (Kawai and Akira, 2010). RNA-polymerase III also transcribesB-DNA into 5′-ppp RNA, which then activates type I IFN transcriptionthrough RIG-I (Chiu et al., 2009). Once phosphorylated, thesetranscription factors help drive the expression of all genes of the typeI IFN family, thereby amplifying type I IFN production. Many cytosolicDNA sensors have been reported to recognize intracellular pathogenicDNAs. See e.g. FIG. 26, excerpted from Xia et al., “DNA sensorcGAS-mediated immune recognition,” Protein Cell, 7(11): 777-791 (2016)).

For example, DDX41 (Zhang et al., 2011b), IFI16 (Orzalli et al., 2012;Unterholzner et al., 2010) and DAI (Takaoka et al., 2007) detect doublestranded DNAs (dsDNAs) and activate the STING-TBK1-IRF3 pathway. LRRFIP1binds dsDNA and triggers IRF3 activation through β-catenin (Yang et al.,2010). DHX9 and DHX36 associate with dsDNA and lead to NFκB activationthrough MyD88 (Kim et al., 2010). Ku70 binds dsDNA to induce type Iinterferon (IFN) through activation of IRF1 and IRF7 (Zhang et al.,2011a). AIM2 interacts with dsDNA and activates inflammasomes byrecruiting ASC and pro-caspase-1 (Burckstummer et al., 2009;Fernandes-Alnemri et al., 2009; Hornung et al., 2009). Of note, Sox2 isexpressed in the cytosol of neutrophils and activates the Tab2/TAK1complex upon binding to dsDNA in a sequence-dependent manner (Xia etal., 2015).

v. DNAs (dsDNAs) Longer than 36 bp and DNA-RNA Hybrids

cGAS is a DNA sensor that recognizes cytoplasmic DNA (Ablasser et al.,2013a; Ablasser et al., 2013b; Gao et al., 2013a; Li et al., 2013b;Schoggins et al., 2014; Sun et al., 2013; Wu et al., 2013). Doublestranded DNAs (dsDNAs) longer than 36 bp are optimal for cGAS activation(Gao et al., 2013b). Post-DNA binding, cGAS undergoes a conformationalchange that allows ATP and GTP to come into the catalytic pocket,leading to the synthesis of cGAMP a strong activator of the STING-TBK1axis (Civril et al., 2013; Gao et al., 2013b; Kranzusch et al., 2013; Wuet al., 2013; Zhang et al., 2014). cGAS can be activated by dsDNAs andDNA-RNA hybrids (Mankan et al., 2014).

vi. Bacterial Second Messenger Cyclic-Di-GMP

Bacterial second messenger cyclic-di-GMP potently induces type I IFN viaa mechanism that is independent of DAI or other known cytoplasmicreceptors but requires TBK1 and IRF3 (McWhirter et al., 2009).

vii. TLR3, TLR4, TLR7, TLR8 and TLR9 Agonists

In some cell types, e.g., macrophages and DCs, type I IFN is produced inresponse to triggering of the transmembrane receptors Toll-like receptor3 (TLR3) and TLR4 by dsRNA and lipopolysaccharide, respectively. TLR3and TLR4 signal through the adaptor molecule TRIF, which associates withTBK1 and activates IRF3 (Kawai and Akira, 2010).

Natural IFN-producing cells, plasmacytoid DCs (pDCs) (Colonna et al.,2004) preferentially express the intracellular endosomal receptors TLR7and TLR9, allowing them to respond to single-stranded RNA and DNAviruses, respectively, by triggering signal transduction through theadaptor protein MyD88 (Colonna et al., 2004). These receptors areefficient in inducing type I IFN only in pDCs because these cellsconstitutively express IRF7 and IRF8, and the MyD88-IRF7 complexundergoes a spatiotemporal regulation upon TLR ligation such that it isretained in the endosomal compartment, where it induces type I IFNproduction (Colonna et al., 2004).

The TLR4 agonist glucopyranosyl lipid adjuvant (GLA) is being testedalone or in combination with anti-PD-1 mAb [Immune Design 2016] (J.Meulen and S. Brady, “Immune Design,” Hum. Vaccin. Immunother.,13(1):15-16 (2017)). The TLR3 agonist Poly-ICLC (Hiltonol™) and theTLR7/8 agonist MEDI9197 also are being tested in patients with advancedaccessible solid tumors (MedImmune 2016; Oncovir 201). (“Activating theNatural Host Defense; Hiltonol (Poly-ICLC) and Malignant Brain Tumors,A. Salzar, Oncovir, Inc., www.oncovir.com/id2 (accessed Jul. 11, 2018);and Gupta et al., “Abstract CT091: Safety and pharmacodynamic activityof MEDI9197, a TLR 7/8 agonist, administered intratumorally in subjectswith solid tumors,” Cancer Research, AACR Annual Meeting 2017; Apr. 1-5,2017 (published July 2017)). Intratumoral injection of TLR agonists suchas CpG-rich oligodeoxynucleotides (CpG ODN, PF-3512676) along withlow-dose radiotherapy has shown clinical responses in patients withadvanced non-Hodgkin's lymphoma in a phase I/II clinical study [Dynavax2016]. (Adamus et al., 2018).

viii. STING Agonists

Cyclic dinucleotides (CDNs) [cyclic di-GMP (guanosine 5′-monophosphate),cyclic di-AMP (adenosine 5′-monophosphate), and cyclic GMP-AMP (cGAMP)]are a class of pathogen-associated molecular pattern molecules (PAMPs)that activate the TBK1/IRF3/type 1 interferon signaling axis via thecytoplasmic pattern recognition receptor stimulator of interferon genes(STING).

New STING agonists are being developed to elicit a type I interferonresponse. One major approach involves rational modifications of CDNs toimprove efficiency, which led to the development of syntheticdithio-mixed linkage CDNs (Corrales et al., 2015). One compound (MLRR-S2 CDA or ADU-S100) binds both human and mouse STING, and showed apotent anti-tumor effect in multiple animal models (Corrales et al.,2015). A phase 1 clinical trial of ADU-S100 in patients with cutaneouslyaccessible solid tumors and lymphomas is in progress (Aduro Biotech2016).

An analysis of the 1000 Genome Project database(http://www.1000genomes.org/) identified five human STING variantsincluding the WT allele, the reference (REF) allele (R232H), the HAQallele (R71H, G230A, R293Q), the AQ allele (G230A, R293Q), and the Qallele (R293Q) (Yi et al., 2013).

A rationally designed synthetic CDN agonist, ML RR-S2 CDA, has beendeveloped and exhibits enhanced stability, human STING activation,cellular uptake, and antitumor efficacy, as well as low reactogenicitycompared with the natural STING ligands produced by bacteria or hostcell cGAS (Corrales et al., 2015; Fu et al., 2015).

Rp, Rp (R,R) dithio-substituted diastereomer CDNs were resistant todigestion with phosphodiesterase, stimulated higher expression of IFN-3in cultured human cells, and induced more potent antitumor immunity ascompared with CDNs that did not contain a dithio modification (Corraleset al., 2015; Fu et al., 2015). To increase affinity for human STING, MLRR-S2 CDA contains a noncanonical structure defined by a phosphatebridge with one 2′-5′ and one 3′-5′ mixed phosphodiester linkages (2′,3′CDNs). The 2′,3′ mixed linkage structure confers increased STING bindingaffinity (Gao et al., 2013b) and is also found in endogenous cGAMPproduced by eukaryotic cGAS. ML RR-S2 CDA was shown to broadly activateall known human STING alleles in a HEK293T cellular STING signalingassay and induced dose-dependent expression of IFN-β in human peripheralblood monocytes (PBMCs) isolated from multiple donors with differentSTING genotypes, including a donor homozygous for the REF allele, whichis known to be refractory to signaling induced by bacterial 3′,3′ CDNs(Corrales et al., 2015; Fu et al., 2015).

C. Type II Interferon Agonists

The present compositions and methods can include a type II IFN agonist,i.e., an agent that increases the level (e.g., the activity orexpression level) of type II interferons. The class of type IIinterferons (IFNs) currently includes a member, called IFN-γ (gamma).Mature IFN-γ is an anti-parallel homodimer, which binds to the IFN-γreceptor (IFNGR) complex to elicit a signal within its target cell.IFNGR is made up of two subunits each of molecules designated IFNGR1 andIFNGR2. IFN-γ is involved in the regulation of the immune andinflammatory responses; in humans, there is only one type ofinterferon-gamma. It is produced in activated T cells and natural killercells. IFN-γ potentiates the effects of type I IFNs. IFN-γ released byTh1 cells recruits leukocytes to a site of infection, resulting inincreased inflammation. It also stimulates macrophages to kill bacteriathat have been engulfed. IFN-γ released by Th1 cells also is importantin regulating the Th2 response. As IFN-γ is vitally implicated in theregulation of immune response, and its production can lead to autoimmunedisorders.

Thus, one embodiment of the invention encompasses compositions thatcomprise a minicell comprising a type II IFN agonist. Although minicellsare derived from bacteria, the minicells by themselves do not activatetype II interferon responses in human patients. See Example 15. Thepresent inventors discovered that the addition of IFN gamma augmentedthe anti-tumor efficacy of EGFR-targeted EDVs loaded with doxorubicinand caused tumor regression in xenograft models. See Example 13.Furthermore, a composition comprising (i) EFGR targeted minicells loadedwith the supertoxic chemotherapy drug PNU-159682, (ii) non-targetedminicells loaded with double stranded DNA comprising 60 nucleotides, and(iii) minicells comprising the IFN gamma product Imukin waswell-tolerated and induced anti-cancer effects in dogs suffering fromlate-stage endogenous tumors. See Example 14.

Type II IFNs play an important role in anti-tumor immunity by activatingcytotoxic T cells. See, e.g., Chikuma et al., 2017. IFN gamma cytokinesare released by innate Natural Killer cells upon binding of naturalantigen, but glycosphingolipid compounds can function as potentactivators of both innate and acquired immune responses. The presentinventors discovered that exposure to a glycosphingolipid induces apotent cytokine response by innate natural killer T (iNKT) cells,including the type II interferon, IFN-γ, and a number of Interleukins(Th1-, Th2-, and/or Th17-type cytokines). See, e.g., Carreno et al.,2016. iNKT cells then induce DC maturation and display T cellhelper-like functions that result in the development of cytotoxic T cellresponses.

Examples of glycosphingolips useful to induce a IFN type II response aredescribed herein and include C-glycosidific form of α-galactosylceramide(α-C-GalCer), α-galactosylceramide (α-GalCer), 12 carbon acyl form ofgalactosylceramide (β-GalCer), β-D-glucopyranosylceramide (β-GlcCer),1,2-Diacyl-3-0-galactosyl-sn-glycerol (BbGL-II), diacylglycerolcontaining glycolipids (Glc-DAG-s2), ganglioside (GD3),gangliotriaosylceramide (Gg3Cer), glycosylphosphatidylinositol (GPI),α-glucuronosylceramide (GSL-1 or GSL-4), isoglobotrihexosylceramide(iGb3), lipophosphoglycan (LPG), lyosphosphatidylcholine (LPC),α-galactosylceramide analog (OCH), and threitolceramide. In a particularembodiment the minicell disclosed herein comprises α-galactosylceramide(α-GalCer) as a type II IFN agonist.

α-GC, an INF type II agonist is known to stimulate the immune systemthrough activation of a type of white blood cell known as natural killerT cell (NKT cell) (Birkholz et al 2015). Knowing that minicells wereable to facilitate presentation of α-GC on target cells, as furtherdiscussed in Example 17, Applicant moved to investigate minicellfacilitated immune activation using minicell_(α-GC) could complementtreatment consisting of minicell facilitated delivery ofchemotherapeutic drug.

As shown in Example 18, Applicant discovered that tumor containing micethat were administered minicells containing the chemotherapeuticdoxorubicin (^(Ep)minicell_(Dox)) and minicells containing α-GC(minicell_(α-Gc)) displayed a marked halt in tumor progression over miceadministered only ^(Ep)minicell_(Dox). These observations indicated thatminicell compositions excluding the INF type I agonist and insteadincorporating an INF type II agonist, are effective at treating tumorsin mice.

The minicell can deliver type II IFN agonists directly to cells of theimmune system, with a view to enhancing iNKT cell activation and type IIinterferon IFN-γ production in vivo. Alternatively, non-targeted EDVsare taken up by phagocytic cells of the immune system, where they arebroken down in endosomes, and aGC is presented to iNKT cells for immuneactivation. Accordingly, in some embodiments the minicell providestargeted delivery of type II interferon agonists. In other embodiments,the composition disclosed herein comprises a non-targeted minicellcomprising a type II interferon agonist.

IFN-γ production is controlled by cytokines secreted by antigenpresenting cells (APCs), most notably interleukin (IL)-12 and IL-18.These cytokines serve as a bridge to link infection with IFN-γproduction in the innate immune response. Macrophage recognition of manypathogens induces secretion of IL-12 and chemokines. These chemokinesattract NK cells to the site of inflammation, and IL-12 promotes IFN-γsynthesis in these cells. In macrophages, natural killer cells and Tcells, the combination of IL-12 and IL-18 stimulation further increasesIFN-γ production. Accordingly, any of these proteins or theircombinations are suitable agents for the purpose of this disclosure.

Negative regulators of IFN-gamma production include IL-4, IL-10,transforming growth factor β and glucocorticoids. Proteins or nucleicacids that inhibit these factors will be able to stimulate theproduction of IFN-γ.

Also suitable for use in this context are polynucleotides that encodeIFN-γ or genes that activate the production and/or the secretion ofIFN-γ.

The agent that increases the level of IFN-γ may also be a viral vaccine.A number of viral vaccines are available that can induce IFN-γproduction without causing infection or other types of adverse effects.Illustrative of this class of viral-vaccine agent is a flu (influenza)vaccine.

The data show that the serum concentration of IFN-γ required foreffectively activating host immune response to tumor cells is low whenthe patient also receives administration of drug-loaded, bispecificantibody-targeted minicells or killed bacterial cells. Thus, in oneaspect the inventive methodology results in increase of serum IFN-γconcentration that is not higher than about 30,000 pg/mL. In anotheraspect, the serum IFN-γ concentration is increased to not higher thanabout 5000 pg/mL, 1000 pg/mL, 900 pg/mL, 800 pg/mL, 700 pg/mL, 600pg/mL, 500 pg/mL, 400 pg/mL, 300 pg/mL, 200 pg/mL, or 100 pg/mL. In afurther aspect, the resulting serum IFN-gamma concentration is at leastabout 10 pg/mL, or at least about 20 pg/mL, 30 pg/mL, 40 pg/mL, 50pg/mL, 60 pg/mL, 70 pg/mL, 80 pg/mL, 90 pg/mL, 100 pg/mL, 150 pg/mL, 200pg/mL, 300 pg/mL, 400 pg/mL or 500 pg/mL.

Pursuant to some aspects, the agent is an IFN-γ protein or an engineeredprotein or analog. In some aspects, the administration achieves fromabout 0.02 ng to 1 microgram of IFN-γ per ml of host blood. In oneaspect, the achieved IFN-gamma concentration in the host blood is fromabout 0.1 ng to about 500 ng per ml, from about 0.2 ng to about 200 ngper ml, from about 0.5 ng to about 100 ng per ml, from about 1 ng toabout 50 ng per ml, or from about 2 ng to about 20 ng per ml.

III. Intact Bacterially-Derived Minicells

The term “minicell” is used here to denote a derivative of a bacterialcell that lacks chromosomes (“chromosome-free”) and is engendered by adisturbance in the coordination, during binary fission, of cell divisionwith DNA segregation. Minicells are distinct from other small vesicles,such as so-called “membrane blebs” (about 0.2 μm or less in size), whichare generated and released spontaneously in certain situations but whichare not due to specific genetic rearrangements or episomal geneexpression. By the same token, intact minicells are distinct frombacterial ghosts, which are not generated due to specific geneticrearrangements or episomal gene expression. Bacterially derivedminicells employed in this disclosure are fully intact and thus aredistinguished from other chromosome-free forms of bacterial cellularderivatives characterized by an outer or defining membrane that isdisrupted or degraded, even removed. See U.S. Pat. No. 7,183,105 at col.111, lines 54 et seq. The intact membrane that characterizes theminicells of the present disclosure allows retention of the therapeuticpayload within the minicell until the payload is released, post-uptake,within a tumor cell.

Minicell or EDVs are anucleate, non-living nanoparticles produced as aresult of inactivating the genes that control normal bacterial celldivision, thereby de-repressing polar sites of cell. Ma et al., 2004.The de-repression means that the bacteria divide in the centre as wellas at the poles; the polar division resulting in minicells which theinventors of the present disclosure have shown can function asleak-resistant, micro-reservoir carriers that allow efficient packagingof a range of different chemotherapeutic drugs. Moreover, in contrast tocurrent stealth liposomal drug carriers like DOXIL (liposomaldoxorubicin), for example, that can package only ˜14,000 molecules perparticle (Park et al., Breast Cancer Res., 4(3): 95-99 (2002), or “armedantibodies,” which can carry fewer than 5 drug molecules, EDVs canreadily accommodate payloads of up to 1 million drug molecules. Further,EDVs can be targeted to over-expressed receptors on the surface ofcancer cells using bispecific antibodies, see section D infra, whichallows highly significant tumor growth-inhibition and/or regression,both in vitro and in vivo.

The minicells employed in the present invention can be prepared frombacterial cells, such as E. coli and S. typhymurium. Prokaryoticchromosomal replication is linked to normal binary fission, whichinvolves mid-cell septum formation. In E. coli, for example, mutation ofmin genes, such as minCD, can remove the inhibition of septum formationat the cell poles during cell division, resulting in production of anormal daughter cell and an chromosome-less minicell. See de Boer etal., J. Bacteriol., 174: 63-70 (1992); Raskin & de Boer, J. Bacteriol.,181: 6419-s24 (1999); Hu & Lutkenhaus, Mol. Microbio., 34: 82-90 (1999);Harry, Mol. Microbiol., 40: 795-803 (2001).

In addition to min operon mutations, chromosome-less minicells also aregenerated following a range of other genetic rearrangements or mutationsthat affect septum formation, for example, in the divIVB 1 in B.subtilis. See Reeve and Cornett, J. Virol., 15: 1308-16 (1975).Minicells also can be formed following a perturbation in the levels ofgene expression of proteins involved in cell division/chromosomesegregation. For instance, over-expression of minE leads to polardivision and production of minicells. Similarly, chromosome-lessminicells can result from defects in chromosome segregation, e.g., thesmc mutation in Bacillus subtilis (Britton et al., Genes Dev., 12:1254-9 (1998)), the spoOJ deletion in B. subtilis (Ireton et al., J.Bacteriol., 176: 5320-29 (1994)), the mukB mutation in E. coli (Hiragaet aL, J. Bacteriol., 171: 1496-1505 (1989)), and the parC mutation inE. coli (Stewart and D'Ari, J. Bacteriol., 174: 4513-6 (1992)). Further,CafA can enhance the rate of cell division and/or inhibit chromosomepartitioning after replication (Okada et al., J. Bacteriol., 176: 917-22(1994)), resulting in formation of chained cells and chromosome-lessminicells.

Accordingly, minicells can be prepared for the present disclosure fromany bacterial cell, be it of Gram-positive or Gram-negative origin dueto the conserved nature of bacterial cell division in these bacteria.Furthermore, the minicells used in the disclosure should possess intactcell walls (i.e., are “intact minicells”), as noted above, and should bedistinguished over and separated from other small vesicles, such asmembrane blebs, which are not attributable to specific geneticrearrangements or episomal gene expression.

In a given embodiment, the parental (source) bacteria for the minicellscan be Gram positive, or they can be Gram negative. In one aspect, theparental bacteria are one or more selected from Terra-/Glidobacteria(BV1), Proteobacteria (BV2), BV4 including Spirochaetes,Sphingobacteria, and Planctobacteria. Pursuant to another aspect, thebacteria are one or more selected from Firmicutes (BV3) such as Bacilli,Clostridia or Tenericutes/Mollicutes, or Actinobacteria (BV5) such asActinomycetales or Bifidobacteriales.

Pursuant to the invention, killed bacterial cells are non-livingprokaryotic cells of bacteria, cyanobateria, eubacteria andarchaebacteria, as defined in the 2nd edition of Bergey's Manual OfSystematic Biology. Such cells are deemed to be “intact” if they possessan intact cell wall and/or cell membrane and contain genetic material(nucleic acid) that is endogenous to the bacterial species. Methods ofpreparing killed bacterial cells are described, for instance, in U.S.patent application publication No. 2008/0038296, the contents of whichare incorporated herein by reference.

In yet a further aspect, the bacteria are one or more selected fromEobacteria (Chloroflexi, Deinococcus-Thermus), Cyanobacteria,Thermodesulfobacteria, thermophiles (Aquificae, Thermotogae), Alpha,Beta, Gamma (Enterobacteriaceae), Delta or Epsilon Proteobacteria,Spirochaetes, Fibrobacteres, Chlorobi/Bacteroidetes,Chlamydiae/Verrucomicrobia, Planctomycetes, Acidobacteria,Chrysiogenetes, Deferribacteres, Fusobacteria, Gemmatimonadetes,Nitrospirae, Synergistetes, Dictyoglomi, Lentisphaerae Bacillales,Bacillaceae, Listeriaceae, Staphylococcaceae, Lactobacillales,Enterococcaceae, Lactobacillaceae, Leuconostocaceae, Streptococcaceae,Clostridiales, Halanaerobiales, Thermoanaerobacterales, Mycoplasmatales,Entomoplasmatales, Anaeroplasmatales, Acholeplasmatales,Haloplasmatales, Actinomycineae, Actinomycetaceae, Corynebacterineae,Nocardiaceae, Corynebacteriaceae, Frankineae, Frankiaceae,Micrococcineae, Brevibacteriaceae, and Bifidobacteriaceae.

For pharmaceutical use, a composition of the disclosure should compriseminicells or killed bacterial cells that are isolated as thoroughly aspossible from immunogenic components and other toxic contaminants.Methodology for purifying bacterially derived minicells to remove freeendotoxin and parent bacterial cells are described, for example, in WO2004/113507, which is incorporated by reference herein in its entirety.Briefly, the purification process achieves removal of (a) smallervesicles, such as membrane blebs, which are generally smaller than 0.2 min size, (b) free endotoxins released from cell membranes, and (c)parental bacteria, whether live or dead, and their debris, which alsoare sources of free endotoxins. Such removal can be implemented with,inter alia, a 0.2 m filter to remove smaller vesicles and cell debris, a0.45 m filter to remove parental cells following induction of theparental cells to form filaments, antibiotics to kill live bacterialcells, and antibodies against free endotoxins.

Underlying the purification procedure is a discovery by the presentinventors that, despite the difference of their bacterial sources, allintact minicells are approximately 400 nm in size, i.e., larger thanmembrane blebs and other smaller vesicles and yet smaller than parentalbacteria. Size determination for minicells can be accomplished by usingsolid-state, such as electron microscopy, or by liquid-based techniques,e.g., dynamic light scattering. The size value yielded by each suchtechnique can have an error range, and the values can differ somewhatbetween techniques. Thus, the size of minicells in a dried state can bemeasured via electron microscopy as approximately 400 nm±50 nm. Dynamiclight scattering can measure the same minicells to be approximately 500nm±50 nm in size. Also, drug-packaged, ligand-targeted minicells can bemeasured, again using dynamic light scattering, to be approximately 400nm to 600 nm±50 nm.

This scatter of size values is readily accommodated in practice, e.g.,for purposes of isolating minicells from immunogenic components andother toxic contaminants, as described above. That is, an intact,bacterially derived minicell is characterized by cytoplasm surrounded bya rigid membrane, which gives the minicell a rigid, spherical structure.This structure is evident in transmission-electron micrographs, in whichminicell diameter is measured, across the minicell, between the outerlimits of the rigid membrane. This measurement provides theabove-mentioned size value of 400 nm±50 nm.

Another structural element of a killed bacterial cells or a minicellderived from Gram-negative bacteria is the O-polysaccharide component oflipopolysaccharide (LPS), which is embedded in the outer membrane viathe lipid A anchor. The component is a chain of repeatcarbohydrate-residue units, with as many as 70 to 100 repeat units offour to five sugars per repeat unit of the chain. Because these chainsare not rigid, in a liquid environment, as in vivo, they can adopt awaving, flexible structure that gives the general appearance of seaweedin a coral sea environment; i.e., the chains move with the liquid whileremaining anchored to the minicell membrane.

Influenced by the O-polysaccharide component, dynamic light scatteringcan provide a value for minicell size of about 500 nm to about 600 nm,as noted above. Nevertheless, minicells from Gram-negative andGram-positive bacteria alike readily pass through a 0.45 μm filter,which substantiates an effective minicell size of 400 nm±50 nm. Theabove-mentioned scatter in sizes is encompassed by the present inventionand, in particular, is denoted by the qualifier “approximately” in thephrase “approximately 400 nm in size” and the like.

In relation to toxic contaminants, a composition of the disclosurepreferably comprises less than about 350 EU free endotoxin. Illustrativein this regard are levels of free endotoxin of about 250 EU or less,about 200 EU or less, about 150 EU or less, about 100 EU or less, about90 EU or less, about 80 EU or less, about 70 EU or less, about 60 EU orless, about 50 EU or less, about 40 EU or less, about 30 EU or less,about 20 EU or less, about 15 EU or less, about 10 EU or less, about 9EU or less, about 8 EU or less, about 7 EU or less, about 6 EU or less,about 5 EU or less, about 4 EU or less, about 3 EU or less, about 2 EUor less, about 1 EU or less, about 0.9 EU or less, about 0.8 EU or less,about 0.7 EU or less, about 0.6 EU or less, about 0.5 EU or less, about0.4 EU or less, about 0.3 EU or less, about 0.2 EU or less, about 0.1 EUor less, about 0.05 EU or less, or about 0.01 EU or less.

A composition of the disclosure also can comprise at least about 10⁹minicells or killed bacterial cells, e.g., at least about 1×10⁹, atleast about 2×10⁹, at least about 5×10⁹, or at least 8×10⁹. In someembodiments, the composition comprises no more than about 10¹¹ minicellsor killed bacterial cells, e.g., no more than about 1×10¹¹ or no morethan about 9×10¹⁰, or no more than about 8×10¹⁰.

IV. Loading Active Agents into Minicells or Killed Bacterial Cells

Active agents or anti-neoplastic agents, such as small molecular drugs,proteins and functional nucleic acids can be packaged into minicellsdirectly by co-incubating a plurality of intact minicells with theactive agent in a buffer. The buffer composition can be varied, as afunction of conditions well known in this field, to optimize the loadingof the active agent in the intact minicells. The buffer also may bevaried in dependence on the agent (e.g., dependent upon the nucleotidesequence or the length of the nucleic acid to be loaded in the minicellsin the case of a nucleic acid payload). An exemplary buffer suitable forloading includes, but is not limited to, phosphate buffered saline(PBS). Once packaged, the active agent remains inside the minicell andis protected from degradation. Prolonged incubation studies withsiRNA-packaged minicells incubated in sterile saline have shown, forexample, no leakage of siRNAs.

Active agents such as functional nucleic acids or proteins that can beencoded for by a nucleic acid, can be introduced into minicells bytransforming into the parental bacterial cell a vector, such as aplasmid, that encodes the active agents. When a minicell is formed fromthe parental bacterial cell, the minicell retains certain copies of theplasmid and/or the expression product, the anti-neoplastic agent. Moredetails of packaging and expression product into a minicell is providedin WO 03/033519, the contents of which are incorporated into the presentdisclosure in its entirety by reference.

Data presented in WO 03/033519 demonstrated, for example, thatrecombinant minicells carrying mammalian gene expression plasmids can bedelivered to phagocytic cells and to non-phagocytic cells. WO 03/033519also described the genetic transformation of minicell-producing parentbacterial strains with heterologous nucleic acids carried onepisomally-replicating plasmid DNAs. Upon separation of parent bacteriaand minicells, some of the episomal DNA segregated into the minicells.The resulting recombinant minicells were readily engulfed by mammalianphagocytic cells and became degraded within intracellularphagolysosomes. Moreover, some of the recombinant DNA escaped thephagolysosomal membrane and was transported to the mammalian cellnucleus, where the recombinant genes were expressed.

In other embodiments, multiple nucleic acids directed to different mRNAtargets can be packaged in the same minicell. Such an approach can beused to combat drug resistance and apoptosis resistance. For instance,cancer patients routinely exhibit resistance to chemotherapeutic drugs.Such resistance can be mediated by over-expression of genes such asmulti-drug resistance (MDR) pumps and anti-apoptotic genes, amongothers. To combat this resistance, minicells can be packaged withtherapeutically significant concentrations of functional nucleic acid toMDR-associated genes and administered to a patient before chemotherapy.Furthermore, packaging into the same minicell multiple functionalnucleic acid directed to different mRNA targets can enhance therapeuticsuccess since most molecular targets are subject to mutations and havemultiple alleles. More details of directly packaging a nucleic acid intoa minicell is provided in WO 2009/027830, the contents of which areincorporated into the present disclosure in its entirety by reference.

Small molecule drugs, whether hydrophilic or hydrophobic, can bepackaged in minicells by creating a concentration gradient of the drugbetween an extracellular medium comprising minicells and the minicellcytoplasm. When the extracellular medium comprises a higher drugconcentration than the minicell cytoplasm, the drug naturally moves downthis concentration gradient, into the minicell cytoplasm. When theconcentration gradient is reversed, however, the drug does not move outof the minicells. More details of the drug loading process and itssurprising nature are found, for instance, in U.S. Patent ApplicationPublication No. 2008/0051469, the contents of which are specificallyincorporated by reference.

To load minicells with drugs that normally are not water soluble, thedrugs initially can be dissolved in an appropriate solvent. For example,paclitaxel can be dissolved in a 1:1 blend of ethanol and cremophore EL(polyethoxylated castor oil), followed by a dilution in PBS to achieve asolution of paclitaxel that is partly diluted in aqueous media andcarries minimal amounts of the organic solvent to ensure that the drugremains in solution. Minicells can be incubated in this final medium fordrug loading. Thus, the inventors discovered that even hydrophobic drugscan diffuse into the cytoplasm or the membrane of minicells to achieve ahigh and therapeutically significant cytoplasmic drug load. This isunexpected because the minicell membrane is composed of a hydrophobicphospholipid bilayer, which would be expected to prevent diffusion ofhydrophobic molecules into the cytoplasm. The loading into minicells ofa diversity of representative small molecule drugs has been shown,illustrating different sizes and chemical properties: doxorubicin,paclitaxel, fluoro-paclitaxel, cisplatin, vinblastine, monsatrol,thymidylate synthase (TS) inhibitor OSI-7904, irinotecan,5-fluorouracil, gemcitabine, and carboplatin. Across the board,moreover, the resultant, small molecule drug-packaged minicells showsignificant anti-tumor efficacy, in vitro and in vivo.

V. Targeting Minicells to Specific Mammalian Cells and Tumors

The inventors discovered that blood vessels around tumor cells display aloss of integrity; that is, the vessels have large fenestrations and are“leaky,” even in the blood brain barrier (BBB) environment. When cancercells establish, they secrete substances that promote the formation ofnew blood vessels—a process called angiogenesis. These blood vesselsgrow quickly and, unlike normal blood vessels, they are leaky with“holes” (fenestrations) ranging from 50 nm to 1.2 m (hyperpermeablevasculature). Drug delivery particles such as liposomes are currentlybelieved to effect tumor-targeting by a passive process involvingextravasation from the leaky vasculature that supports the tumormicroenvironment. Hobbs et al., 1998. Although it has been shown thatthe abnormal tumor microenvironment is characterised by interstitialhypertension, and that this phenomenon may limit access of anti-cancerantibody therapeutics, this does not appear to be an absolute barrier asis exemplified by immunoliposomes (Nielsen et al, 2002) and antibodyconjugated to Quantum Dots (Gao et aL, 2004). This phenomenon also holdstrue for the EDV which has the added advantage of carrying aspecifically directed tumor antibody. Following IV injection the EDVextravasates into the tumor microenvironment and this is followed byactive targeting via cancer cell-surface receptor engagement andendocytosis. In contrast to conventional understanding, therefore,particles that are as large as minicells, i.e., much larger than theabove-discussed consensus pore size limitations of the BBB, neverthelessare smaller than the fenestrations in the walls of the leaky bloodvessel; hence, they can extravasate passively through thesefenestrations and into the tumor microenvironment.

Upon entering the tumor microenvironment, minicells are able to triggerreceptor-mediated internalization by the host tumor cells and to betaken up by them. Thus, a minicell that is packaged with ananti-neoplastic agent will release the agent into the cytoplasm of thetumor cell, killing it.

Pursuant to a further aspect of this disclosure, the minicells or killedbacterial cells of a composition, as described above, are directed to atarget mammalian tumor cell via a ligand. In some embodiments the ligandis “bispecific.” That is, the ligand displays a specificity for bothminicell and mammalian (tumor) cell components, such that it causes agiven vesicle to bind to the target cell, whereby the latter engulfs theformer. Use of bispecific ligands to target a minicell to a tumor cellis further described in WO 05/056749 and WO 05/079854, and use ofbispecific ligands to target a killed bacterial cell to a tumor cell isfurther described in U.S. Pat. No. 8,591,862, the respective contents ofwhich are incorporated here by reference in its entirety. Once such aligand is attached to a vesicle, the unoccupied specificity(“monospecificity”) of the ligand pertains until it interacts with thetarget (tumor) mammalian cell. A number of tumor targeting ligands areknown in the art (Hong et al., 2011; Hoelder et al., 2012; Galluzzi etal., 2013). Several peptides, such as somatostatin (SST) peptide,vasoactive intestinal peptide (VIP), Arg-Gly-Asp (RGD) peptide, andbombesin/gastrin-releasing peptide (BBN/GRP), have been successfullycharacterized for tumor receptor imaging (De Jong et al., 2009; Tweedle,2009; Schottelius and Wester 2009; Igarashi et al., 2011; Laverman etal., 2012).

Tumor-targeting peptide sequences can be selected mainly in threedifferent ways: (1) derivatization from natural proteins (Nagpal et al.,2011); (2) chemical synthesis and structure-based rational engineering(Andersson et al., 2000; Merrifield, 2006); and (3) screening of peptidelibraries (Gray and Brown 2013). Among the methods, phage displaytechnology is a conventional but most widely used method with manyadvantages such as ease of handling and large numbers of differentpeptides can be screened effectively (Deutscher, 2010).

Receptors that are overexpressed on tumor cells rather than on normalcells are excellent candidates for in vivo tumor imaging. To date, manytumor targeting peptides and their analogs have been identified asdescribed below.

Arg-Gly-Asp (RGD) peptide—RGD specifically binds to integrin receptors(Ruoslahti, 1996). Integrins constitute two subunits (α and β subunits).The integrin family, especially α_(v)β₃, is associated with tumorangiogenesis and metastasis. They are overexpressed on endothelial cellsduring angiogenesis, but barely detectable in most normal organs.Therefore, they are widely used for diagnostic imaging.

Bombesin (BBN)/gastrin-releasing peptide (GRP)—Amphibian BBNs and theirrelated peptides consist of a family of neuropeptides exhibiting variousphysiological effects such as exocrine and endocrine secretions,thermoregulation, sucrose regulations as well as cell growth(Ohki-Hamazaki et al., 2005). The bombesin-like peptide receptors have4-subtypes: the neuromedin B receptor, the bombesin 3 receptor, the GRPreceptor, and the bombesin 4 receptor. These receptors are overexpressedin many tumors such as breast cancer, ovarian cancer andgastrointestinal stromal tumors.

Cholecystokinin (CCK)/gastrin peptide—CCK and gastrin are structurallyand functionally similar peptides that exert a variety of physiologicalactions in the gastrointestinal tract as well as the central nervoussystem (Matsuno et al., 1997). Three types of receptors for CCK (CCK1,CCK2 and CCK2i4sv have been identified, which all belong to thesuperfamily of GPCRs. Among them, CCK2/gastrin receptors have beenfrequently found in human cancers such as stromal ovarian cancers andastrocytomas.

α-Melanocyte-stimulating hormone (α-MSH)-α-MSHs are lineartridecapeptides, mainly responsible for skin pigmentation regulation(Singh and Mukhopadhyay, 2014). α-MSHs and their analogs exhibit bindingaffinities to melanocortin-1 receptors (MC-1r) which are expressed inover 80% of human melanoma metastases, and thus, are widely used asvehicles for melanoma-targeted imaging and radiotherapy.

Neuropeptide Y (NPY)—NPY is a 36 amino acid peptide and belongs to thepancreatic polypeptide family (Tatemoto, 2004). NPY receptors areoverexpressed in various tumors including neuroblastomas, sarcomas, andbreast cancers.

Neutrotensin (NT)—NT is a 13 amino acid peptide, targeting NT receptorwhich has been identified in various tumors such as ductal pancreaticadenocarcinomas, small cell lung cancer, and medullary thyroid cancer(Tyler-McMahon et al., 2000). Therefore, it is an attractive candidatefor cancer imaging.

Prostate Specific Membrane Antigen (PSMA)—Prostate cancer cellsoverexpress PSMA on the cell surface (Silver et al., 2007; Ghosh andHeston, 2004; Mhawech-Fauceglia et al., 2007; Santoni et al., 2014).There are several available radiopharmaceuticals that target PSMAincluding [⁶⁸Ga]Ga-PSMA-HBED-CC (also known as [⁶⁸Ga]Ga-PSMA-11 [PET]),a monoclonal antibody (mAb) [¹⁷⁷Lu]Lu/[⁹⁰Y]Y-J591 (therapy),[¹²³I]I-MIP-1072 (planar/SPECT), [¹³¹I]I-MIP-1095 (therapy), and thetheranostic agents PSMA-I&T and DKFZ-PSMA-617 (PSMA-617), which arelabeled with ⁶⁸Ga for PET or with ¹⁷⁷Lu for therapy.

Somatostatin (SST) peptide—SSTs are naturally occurring cyclopeptidehormones with either 14 or 28 amino acids (Weckbecker et al., 2003).They can inhibit the secretion of insulin, glucagon and some otherhormones. Somatostatin receptors (SSTRs; five subtypes SSTR1-SSTR5) areoverexpressed in many tumors including gliomas, neuroendocrine tumorsand breast tumor. Neuroendocrine neoplasia (NEN) of the GEP systemoriginates most frequently from the pancreas, jejunum, ileum, cecum,rectum, appendix, and colon. The common characteristic of all GEP-NEN isthe compound features of endocrine and nerve cells. Well-differentiatedNEN overexpresses somatostatin receptors (SSTRs), especially the SSTR-2subtype.

Substance P—Substance P is an undecapeptide belonging to a family ofneuropeptides known as tachykinins (Strand, 1999). Substance P is aspecific endogenous ligand known for neurokinin 1 receptor (NKIR) whichis found to be expressed on various cancer cells.

T140—T140 is a 14 amino acid peptide with one disulfide bridge and is aninverse agonist of chemokine receptor type 4 (CXCR4) (Burger et al.,2005). Its derivatives are widely used as CXCR4 imaging agents.

Tumor molecular targeted peptide 1 (TMTP1)—TMTP1 is a 5-amino acidpeptide that has been found to specifically bind to highly metastaticcancer cells, especially those from a typical liver micrometastasis(Yang et al., 2008).

Vasoactive intestinal peptide (VIP)—VIP is a neuropeptide with 28 aminoacids (Igarashi et al., 2011). It promotes vasodilation, cell growth andproliferation. Its action is mainly controlled by two receptor subtypes(VPAC1 and VPAC2). A large amount of VIP receptors are expressed on manytumors including adenocarcinomas of the pancreas and neuroendocrinetumors.

The ligand can be attached to the cell membrane of the vesicles byvirtue of the interaction between the ligand and a component on the cellmembrane, such as a polysaccharide, a glycoprotein, or a polypeptide.The expressed ligand is anchored on the surface of a vesicle such thatthe tumor surface component-binding portion of the ligand is exposed sothat the portion can bind the target mammalian cell surface receptorwhen the vesicle and the mammalian tumor cell come into contact.

Alternatively, the ligand can be expressed and displayed by a livingcounterpart of a bacterially derived vesicle, e.g., by the parent cellof a minicell or by a bacterial cell before it becomes a killed cell. Inthis instance the ligand does not require a specificity to the vesicleand only displays a specificity to a component that is characteristic ofmammalian cells. That is, such component need not be unique to tumorcells, per se, or even to the particular kind of tumor cells undertreatment, so long as the tumor cells present the component on theirsurface.

Upon intravenous administration, vesicles accumulate rapidly in thetumor microenvironment. This accumulation, occurring as a function ofthe above-described leaky tumor vasculature, effects delivery ofvesicle-packaged therapeutic payload to cells of the tumor, which theninternalize packaged vesicles.

The inventors have found that this delivery approach is applicable to arange of mammalian tumor cells, including cells that normally arerefractory to specific adhesion and endocytosis of minicells. Forinstance, ligands that comprise an antibody directed at an anti-HER2receptor or anti-EGF receptor can bind minicells to the respectivereceptors on a range of targeted non-phagocytic cells, such as lung,ovarian, brain, breast, prostate, and skin cancer cells.

The binding thus achieved precedes uptake of the vesicles by each typeof non-phagocytic cells. That is, in the context of the presentinvention a suitable target cell presents a cell surface receptor thebinding of which, by a ligand on a vesicle, elicits endocytosis of thatvesicle.

More specifically, the present inventors discovered that the interactionbetween (a) the ligand on a minicell or a killed bacterial cell and (b)a mammalian cell surface receptor can activate an uptake pathway, calledhere a “receptor-mediated endocytosis” (rME) pathway, into thelate-endosomal/lysosomal compartment of the target host cell, such as atumor cell. By this rME pathway, the inventors found, bacteriallyderived vesicles are processed through the early endosome, the lateendosome and the lysosome, resulting in release of their payload intothe cytoplasm of the mammalian host cell. Moreover, a payload that is anucleic acid not only escapes complete degradation in thelate-endosomal/lysosomal compartment but also is expressed by the hostcell.

A tumor targeting ligand for this delivery approach can be “bispecific,”as described above, because it binds to surface components on apayload-carrying vesicle and on a target cell, respectively, and itsinteraction with the latter component leads to uptake of the vesicleinto the rME pathway. In any event, a given target cell surface receptorcan be a candidate for binding by the ligand, pursuant to the invention,if interaction with the component in effect accesses an endocyticpathway that entails a cytosolic internalization from the target cellsurface. Such candidates are readily assessed for suitability in theinvention via an assay in which a cell type that presents on its surfacea candidate component is co-incubated in vitro with minicells carrying aligand that binds the candidate and that also is joined to a fluorescentdye or other marker amenable to detection, e.g., visually via confocalmicroscopy. (An in vitro assay of this sort is described by MacDiarmidet al., 2007b, in the legend to FIG. 3 at page 436.) Thus, an observedinternalization of the marker constitutes a positive indication by suchan assay that the tested target cell surface receptor is suitable forthe present invention.

In accordance with the invention, the ligand can be any polypeptide orpolysaccharide that exhibits the desired specificity or specificities.Preferred ligands are antibodies. In its present use the term “antibody”encompasses an immunoglobulin molecule obtained by in vitro or in vivogeneration of an immunogenic response. Accordingly, the “antibody”category includes monoclonal antibodies and humanized antibodies, suchas single-chain antibody fragments (scFv), bispecific antibodies, etc. Alarge number of different bispecific protein and antibody-based ligandsare known, as evidenced by the review article of Caravella andLugovskoy, Curr. Opin. Chem. Biol., 14: 520-28 (2010), which isincorporated here by reference in its entirety. Antibodies useful inaccordance with the present disclosure can be obtained by knownrecombinant DNA techniques.

By way of non-limiting example, therefore, an antibody that carriesspecificity for a surface component, such as a tumor antigen, can beused to target minicells to cells in a tumor to be treated. Illustrativecell surface receptors in this regard include any of the RTKs epidermalgrowth factor receptor (EGFR), vascular endothelial growth factorreceptor (VEGFR), platelet-derived growth factor receptor (PDGFR) andinsulin-like growth factor receptor (IGFR), each of which is highlyexpressed in several solid tumors, including brain tumors, and folatereceptor, which is overexpressed in some pituitary adenomas. Such abispecific ligand can be targeted as well to mutant or variantreceptors, e.g., the IL-13Rα2 receptor, which is expressed in 50% to 80%of human glioblastoma multiforme tumors, see Wykosky et al., 2008;Jarboe et al., 2007; Debinski et al, 2000; and Okada et al., 1994), butwhich differs from its physiological counterpart IL4R/IL13R, expressedin normal tissues. See Hershey, 2003. Thus, IL13Rα2 is virtually absentfrom normal brain cells. See Debinski and Gibo, 2000. Additionally,tumors that metastasize to the brain may overexpress certain receptors,which also can be suitable targets. For instance, Da Silva et at, 2010,showed that brain metastases of breast cancer expressed all members ofthe HER family of RTKs. HER2 was amplified and overexpressed in 20% ofbrain metastases, EGFR was overexpressed in 21% of brain metastases,HER3 was overexpressed in 60% of brain metastases and HER4 wasoverexpressed in 22% of brain metastases. Interestingly, HER3 expressionwas increased in breast cancer cells residing in the brain.

Illustrative of candidate target cell surface receptors are members ofthe receptor tyrosine kinases or “RKTs,” a family of transmembraneproteins that undergo constitutive internalization (endocytosis) at arate similar to that of other integral membrane proteins. See Goh andSorkin, 2013. The family of RKTs is described by Lemmon andSchlessinger, Cell, 141(7): 1117-134 (2010). Exemplary RTKs are ErbBEGFR, ErbB2, ErbB3, ErbB4 Ins InsR, IGF1R, InsRR PDGF PDGFR.alpha.,PDGFR.beta., CSF1R/Fms, Kit/SCFR, Fit3/Flk2 VEGF VEGFR1/Fit1,VEGFR2/KDR, VEGFR3/Fit4 FGF FGFR1, FGFR2, FGFR3, FGFR4 PTK7 PTK7/CCK4Trk TrkA, TrkB, TrkC Ror Ror1, Ror2 MuSK Met, Ron Ax1, Mer, Tyro3 TieTie1, Tie2 Eph EphA1-8, EphA10, EphB1-4, EphB6 Ret Ryk DDR DDR1, DDR2Ros LMR LMR1, LMR2, LMR3 ALK, LTK STYK1 SuRTK106/STYK1.

Another candidate for suitable target cell surface receptors are thefamily of membrane-associated, high-affinity folate binding proteins(folate receptor), which bind folate and reduced folic acid derivativesand which mediate delivery of tetrahydrofolate to the interior of cells;the family of membrane-bound cytokine receptors that play a role in theinternalization of a cognate cytokine, such as IL13; the surfaceantigens such as CD20, CD33, mesothelin and HM1.24, that are expressedon certain cancer cells and that mediate the internalization of cognatemonoclonal antibodies, e.g., rituximab in the instance of CD20; and thefamily of adhesion receptors (integrins), which are transmembraneglycoproteins that are trafficked through the endosomal pathway and aremajor mediators of cancer cell adhesion. In one embodiment of theinvention, the tumor cell surface receptor comprises an integrin,neuromedin B receptor, bombesin 3 receptor, GRP receptor, bombesin 4receptor, CCK2/gastrin, melanocortin-1 receptor (MC-1r), neuropeptide Y(NPY) receptor, neutrotensin (NT) receptor, prostate specific membraneantigen (PSMA), somatostatin (SST) receptor, neurokinin 1 receptor(NK1R), chemokine receptor type 4 (CXCR4), vasoactive intestinal peptide(VIP), epidermal growth factor receptor (EGFR), vascular endothelialgrowth factor receptor (VEGFR), platelet-derived growth factor receptor(PDGFR), insulin-like growth factor receptor (IGFR), or any combinationthereof.

According to another embodiment of the invention, the cell surfacereceptor is an antigen which is uniquely expressed on a target cell in adisease condition, but which remains either non-expressed, expressed ata low level or non-accessible in a healthy condition. Examples of suchtarget antigens which might be specifically bound by a targeting ligandof the invention may advantageously be selected from EpCAM, CCR5, CD19,HER-2 neu, HER-3, HER-4, EGFR, PSMA, CEA, MUC-1 (mucin), MUC2, MUC3,MUC4, MUC5, MUC5, MUC7, BhcG, Lewis-Y. CD20, CD33, CD30, gangliosideGD3, 9-O-Acetyl-GD3, GM2, Globo H, fucosyl GM1, Poly SA, GD2,Carboanhydrase IX (MN/CA IX), CD44v6, Sonic Hedgehog (Shh), Wue-1,Plasma Cell Antigen, (membrane-bound) IgE, Melanoma Chondroitin SulfateProteoglycan (MCSP), CCR8, TNF-alpha precursor, STEAP, mesothelin, A33Antigen, Prostate Stem Cell Antigen (PSCA), Ly-6; desmoglein 4,E-cadherin neoepitope, Fetal Acetylcholine Receptor, CD25, CA19-9marker, CA-125 marker and Muellerian Inhibitory Substance (MIS) Receptortype II, sTn (sialylated Tn antigen; TAG-72), FAP (fibroblast activationantigen), endosialin, EGFRVIII, LG, SAS and CD63.

VI. Formulations

The invention includes within its scope compositions, or formulations,comprising minicells having as payloads a combination of one or more of(1) an anti-neoplastic agent, (2) a type I IFN agonist, and/or (3) atype II IFN agonist. In compositions comprising all three components,the anti-neoplastic agent, the type I IFN agonist, and the type II IFNagonist can be comprised in one or more minicells. For example: (a) theanti-neoplastic agent, the type I IFN agonist, and the type II IFNagonist can be comprised within the same minicell; (b) theanti-neoplastic agent and the type I IFN agonist can be comprised withina first minicell, and the type II IFN agonist can be comprised within asecond minicell; (c) the anti-neoplastic agent and the type II IFNagonist can be comprised within a first minicell, and the type I IFNagonist can be comprised within a second minicell; or (d) theanti-neoplastic agent can be comprised within a first minicell and thetype I IFN agonist and the type II IFN agonist can be comprised within asecond minicell, or (e) the anti-neoplastic agent can be comprisedwithin a first minicell, the type I IFN agonist can be comprised withina second minicell and the type II IFN agonist can be comprised within athird minicell.

The invention includes within its scope compositions, or formulations,comprising minicells having as payloads a combination of (1) ananti-neoplastic agent and (2) a type I IFN agonist or a type II IFNagonist. In some embodiments, the anti-neoplastic agent and the type IIFN agonist or the INF II agonist can be comprised in one or moreminicells. For example: (a) the anti-neoplastic agent and the type I IFNagonist, can be comprised within the same minicell; (b) theanti-neoplastic agent can be comprised within a first minicell and thetype I IFN agonist can be comprised within a second minicell; (c) theanti-neoplastic agent and the type II IFN agonist can be comprisedwithin the same minicell; or (d) the anti-neoplastic agent can becomprised within a first minicell and the type II IFN agonist can becomprised within a second minicell.

In an exemplary embodiment, the compositions disclosed herein comprisethe anti-neoplastic agent siPlk1, the interferon type I agonist 60merdouble stranded DNA, and/or the interferon type II agonist α-galactosylceramide, wherein the siPlk1, the 60mer double stranded DNA, and theα-galactosyl ceramide are comprised within one or more minicells.

In another exemplary embodiment, the compositions disclosed hereincomprise the anti-neoplastic agent siRRM1, the interferon type I agonist60mer double stranded DNA, and/or the interferon type II agonistα-galactosyl ceramide, wherein the siRRM1, the 60mer double strandedDNA, and the α-galactosyl ceramide are comprised within one or moreminicells.

In another exemplary embodiment, the compositions disclosed hereincomprise the anti-neoplastic agent PNU-159682, the interferon type Iagonist 60mer double stranded DNA, and/or the interferon type II agonistα-galactosyl ceramide, wherein the PNU-159682, the 60mer double strandedDNA, and/or the α-galactosyl ceramide are comprised within one or moreminicells.

The formulations also optionally comprise a bispecific ligand fortargeting the minicell to a target cell. The minicell and ligand may beany of those described herein. Thus, the bispecific ligand of thepresent invention is capable of binding to a surface component of theminicell and to a surface component of a target mammalian cell.

A formulation comprising minicells, drugs and optionally bispecificligands of the present invention (that is, a formulation that includessuch minicells, drugs and ligands with other constituents that do notinterfere unduly with the drug or drug-delivering quality of thecomposition) can be formulated in conventional manner, using one or morepharmaceutically acceptable carriers or excipients.

Formulations or compositions of the disclosure can be presented in unitdosage form, e.g., in ampules or vials, or in multi-dose containers,with or without an added preservative. The formulation can be asolution, a suspension, or an emulsion in oily or aqueous vehicles, andcan contain formulatory agents, such as suspending, stabilizing and/ordispersing agents. A suitable solution is isotonic with the blood of therecipient and is illustrated by saline, Ringer's solution, and dextrosesolution. Alternatively, formulations can be in lyophilized powder form,for reconstitution with a suitable vehicle, e.g., sterile, pyrogen-freewater or physiological saline. The formulations also can be in the formof a depot preparation. Such long-acting formulations can beadministered by implantation (for instance, subcutaneously orintramuscularly) or by intramuscular injection. In some embodiments,administering comprises enteral or parenteral administration. In someembodiments administering comprises administration selected from oral,buccal, sublingual, intranasal, rectal, vaginal, intravenous,intramuscular, and subcutaneous injection.

In some aspects, a minicell-containing composition that includes atherapeutically effective amount of an anti-neoplastic agent isprovided. A “therapeutically effective” amount of an anti-neoplasticagent is a dosage of the agent in question, e.g., a siRNA or asuper-cytotoxic drug that invokes a pharmacological response whenadministered to a subject, in accordance with the present disclosure.

In the context of the present disclosure, therefore, a therapeuticallyeffective amount can be gauged by reference to the prevention oramelioration of the tumor or a symptom of tumor, either in an animalmodel or in a human subject, when minicells carrying a therapeuticpayload are administered, as further described below. An amount thatproves “therapeutically effective amount” in a given instance, for aparticular subject, may not be effective for 100% of subjects similarlytreated for the tumor, even though such dosage is deemed a“therapeutically effective amount” by skilled practitioners. Theappropriate dosage in this regard also will vary as a function, forexample, of the type, stage, and severity of the tumor.

When “therapeutically effective” is used to refer to the number ofminicells in a pharmaceutical composition, the number can be ascertainedbased on what anti-neoplastic agent is packaged into the minicells andthe efficacy of that agent in treating a tumor. The therapeutic effect,in this regard, can be measured with a clinical or pathologicalparameter such as tumor mass. A reduction or reduced increase of tumormass, accordingly, can be used to measure therapeutic effects.

A. Administration Routes

Formulations of the invention can be administered via various routes andto various sites in a mammalian body, to achieve the therapeuticeffect(s) desired, either locally or systemically. Delivery may beaccomplished, for example, by oral administration, by application of theformulation to a body cavity, by inhalation or insufflation, or byparenteral, intramuscular, intravenous, intraportal, intrahepatic,peritoneal, subcutaneous, intratumoral, or intradermal administration.The mode and site of administration is dependent on the location of thetarget cells. For example, tumor metastasis may be more efficientlytreated via intravenous delivery of targeted minicells. Primary ovariancancer may be treated via intraperitoneal delivery of targetedminicells. A combination of routes also may be employed. For example, inmetastatic bladder cancer the cytotoxic drug-loaded andreceptor-targeted minicells may be administered within the bladder aswell as intravenously, and the adjuvant-packaged (receptor-targeted ornon-targeted) minicells along with targeted-drug-packaged minicells maybe administered intravenously. The in situ administration of targeted,drug-packaged minicells may target bladder surface-exposed tumors, whilethe full combination of minicells administered intravenously may targettissue-localized tumors and also elicit the anti-tumor immune response.

B. Purity

Minicells of the invention are substantially free from contaminatingparent bacterial cells. Thus, minicell-comprising formulationspreferably comprise fewer than about 1 contaminating parent bacterialcell per 10⁷ minicells, fewer than about 1 contaminating parentbacterial cell per 10⁸ minicells, fewer than about 1 contaminatingparent bacterial cell per 10⁹ minicells, fewer than about 1contaminating parent bacterial cell per 10¹⁰ minicells, or fewer thanabout 1 contaminating parent bacterial cell per 10¹¹ minicells.

Methods of purifying minicells are known in the art and described inPCT/IB02/04632. One such method combines cross-flow filtration (feedflow is parallel to a membrane surface; Forbes, 1987) and dead-endfiltration (feed flow is perpendicular to the membrane surface).Optionally, the filtration combination can be preceded by a differentialcentrifugation, at low centrifugal force, to remove some portion of thebacterial cells and thereby enrich the supernatant for minicells.

Another purification method employs density gradient centrifugation in abiologically compatible medium. After centrifugation, a minicell band iscollected from the gradient, and, optionally, the minicells aresubjected to further rounds of density gradient centrifugation tomaximize purity. The method may further include a preliminary step ofperforming differential centrifugation on the minicell-containingsample. When performed at low centrifugal force, differentialcentrifugation will remove some portion of parent bacterial cells,thereby enriching the supernatant for minicells.

Particularly effective purification methods exploit bacterialfilamentation to increase minicell purity. Thus a minicell purificationmethod can include the steps of (a) subjecting a sample containingminicells to a condition that induces parent bacterial cells to adopt afilamentous form, followed by (b) filtering the sample to obtain apurified minicell preparation.

Known minicell purification methods also can be combined. One highlyeffective combination of methods is as follows:

Step A: Differential centrifugation of a minicell producing bacterialcell culture. This step, which may be performed at 2,000 g for about 20minutes, removes most parent bacterial cells, while leaving minicells inthe supernatant;

Step B: Density gradient centrifugation using an isotonic and non-toxicdensity gradient medium. This step separates minicells from manycontaminants, including parent bacterial cells, with minimal loss ofminicells. Preferably, this step is repeated within a purificationmethod;

Step C: Cross-flow filtration through a 0.45 μm filter to further reduceparent bacterial cell contamination.

Step D: Stress-induced filamentation of residual parent bacterial cells.This may be accomplished by subjecting the minicell suspension to any ofseveral stress-inducing environmental conditions;

Step E: Antibiotic treatment to kill parent bacterial cells;

Step F: Cross-flow filtration to remove small contaminants, such asmembrane blebs, membrane fragments, bacterial debris, nucleic acids,media components and so forth, and to concentrate the minicells. A 0.2μm filter may be employed to separate minicells from small contaminants,and a 0.1 μm filter may be employed to concentrate minicells;

Step G: Dead-end filtration to eliminate filamentous dead bacterialcells. A 0.45 um filter may be employed for this step; and

Step H: Removal of endotoxin from the minicell preparation. Anti-Lipid Acoated magnetic beads may be employed for this step.

C. Administration Schedules

In general, the formulations disclosed herein may be used at appropriatedosages defined by routine testing, to obtain optimal physiologicaleffect, while minimizing any potential toxicity. The dosage regimen maybe selected in accordance with a variety of factors including age,weight, sex, medical condition of the patient; the severity of thecondition to be treated, the route of administration, and the renal andhepatic function of the patient.

Optimal precision in achieving concentrations of minicell and drugwithin the range that yields maximum efficacy with minimal side effectsmay require a regimen based on the kinetics of the minicell and drugavailability to target sites and target cells. Distribution,equilibrium, and elimination of a minicell or drug may be consideredwhen determining the optimal concentration for a treatment regimen. Thedosages of the minicells and drugs may be adjusted when used incombination, to achieve desired effects.

Moreover, the dosage administration of the formulations may be optimizedusing a pharmacokinetic/pharmacodynamic modeling system. For example,one or more dosage regimens may be chosen and apharmacokinetic/pharmacodynamic model may be used to determine thepharmacokinetic/pharmacodynamic profile of one or more dosage regimens.Next, one of the dosage regimens for administration may be selectedwhich achieves the desired pharmacokinetic/pharmacodynamic responsebased on the particular pharmacokinetic/pharmacodynamic profile. See,e.g., WO 00/67776.

Specifically, the formulations may be administered at least once a weekover the course of several weeks. In one embodiment, the formulationsare administered at least once a week over several weeks to severalmonths.

More specifically, the formulations may be administered at least once aday for about 2, about 3, about 4, about 5, about 6, about 7, about 8,about 9, about 10, about 11, about 12, about 13, about 14, about 15,about 16, about 17, about 18, about 19, about 20, about 21, about 22,about 23, about 24, about 25, about 26, about 27, about 28, about 29,about 30, or about 31 days. Alternatively, the formulations may beadministered about once every day, about once every about 2, about 3,about 4, about 5, about 6, about 7, about 8, about 9, about 10, about11, about 12, about 13, about 14, about 15, about 16, about 17, about18, about 19, about 20, about 21, about 22, about 23, about 24, about25, about 26, about 27, about 28, about 29, about 30 or about 31 days ormore.

The formulations may alternatively be administered about once everyweek, about once every about 2, about 3, about 4, about 5, about 6,about 7, about 8, about 9, about 10, about 11, about 12, about 13, about14, about 15, about 16, about 17, about 18, about 19 or about 20 weeksor more. Alternatively, the formulations may be administered at leastonce a week for about 2, about 3, about 4, about 5, about 6, about 7,about 8, about 9, about 10, about 11, about 12, about 13, about 14,about 15, about 16, about 17, about 18, about 19 or about 20 weeks ormore.

The formulations may alternatively be administered about twice everyweek, about twice every about 2, about 3, about 4, about 5, about 6,about 7, about 8, about 9, about 10, about 11, about 12, about 13, about14, about 15, about 16, about 17, about 18, about 19 or about 20 weeksor more. Alternatively, the formulations may be administered at leastonce a week for about 2, about 3, about 4, about 5, about 6, about 7,about 8, about 9, about 10, about 11, about 12, about 13, about 14,about 15, about 16, about 17, about 18, about 19 or about 20 weeks ormore.

Alternatively, the formulations may be administered about once everymonth, about once every about 2, about 3, about 4, about 5, about 6,about 7, about 8, about 9, about 10, about 11 or about 12 months ormore.

The formulations may be administered in a single daily dose, or thetotal daily dosage may be administered in divided doses of two, three,or four times daily.

In a method in which minicells are administered before a drug,administration of the drug may occur anytime from several minutes toseveral hours after administration of the minicells. The drug mayalternatively be administered anytime from several hours to severaldays, possibly several weeks up to several months after the minicells.

More specifically, the minicells may be administered at least about 1,about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9,about 10, about 11, about 12, about 13, about 14, about 15, about 16,about 17, about 18, about 19, about 20, about 21, about 22, about 23 orabout 24 hours before the drug. Moreover, the minicells may beadministered at least about 1, about 2, about 3, about 4, about 5, about6, about 7, about 8, about 9, about 10, about 11, about 12, about 13,about 14, about 15, about 16, about 17, about 18, about 19, about 20,about 21, about 22, about 23, about 24, about 25, about 26, about 27,about 28, about 29, about 30 or about 31 days before the administrationof the drug. In yet another embodiment, the minicells may beadministered at least about 1, about 2, about 3, about 4, about 5, about6, about 7, about 8, about 9, about 10, about 11, about 12, about 13,about 14, about 15, about 16, about 17, about 18, about 19 or about 20weeks or more before the drug.

In a further embodiment, the minicells may be administered at leastabout 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8,about 9, about 10, about 11 or about 12 months before the drug.

In another embodiment, the minicell is administered after the drug. Theadministration of the minicell may occur anytime from several minutes toseveral hours after administration of the drug. The minicell mayalternatively be administered anytime from several hours to severaldays, possibly several weeks up to several months after the drug.

VII. Methods of Treating Cancer

The compositions described herein may be used to treat a subjectsuffering from a cancer. The method disclosed herein comprisesadministering to the subject an effective amount of a compositionaccording to the invention, comprising at least one anti-neoplasticagent, an interferon type I agonist, an interferon type II agonist, or acombination of an interferon type I agonist and an interferon type IIagonist. The anti-neoplastic agent, the interferon type I agonist, theinterferon type II agonist, or the combination of an interferon type Iagonist and the interferon type II agonist are comprised in one or moreminicells.

In another aspect, the composition used to treat a subject sufferingfrom cancer further comprises a pharmaceutically acceptable carrier.

In another aspect, the methods disclosed herein are useful for treatinga subject suffering from a cancer, wherein the subject is a human, anon-human primate, a dog, a cat, a cow, a sheep, a horse, a rabbit, amouse, or a rat.

In another aspect, the methods disclosed herein are useful for treatinga cancer disease. In some embodiment the cancer comprises a lung cancer,a breast cancer, a brain cancer, a liver cancer, a colon cancer, apancreatic cancer, or a bladder cancer.

In some embodiments, the cancer comprises an acute lymphoblasticleukemia; acute myeloid leukemia; adrenocortical carcinoma; AIDS-relatedcancers; AIDS-related lymphoma; anal cancer; appendix cancer;astrocytomas; atypical teratoid/rhabdoid tumor; basal cell carcinoma;bladder cancer; brain stem glioma; brain tumor (including brain stemglioma, central nervous system atypical teratoid/rhabdoid tumor, centralnervous system embryonal tumors, astrocytomas, craniopharyngioma,ependymoblastoma, ependymoma, medulloblastoma, medulloepithelioma,pineal parenchymal tumors of intermediate differentiation,supratentorial primitive neuroectodermal tumors and pineoblastoma);breast cancer; bronchial tumors; Burkitt lymphoma; cancer of unknownprimary site; carcinoid tumor; carcinoma of unknown primary site;central nervous system atypical teratoid/rhabdoid tumor; central nervoussystem embryonal tumors; cervical cancer; childhood cancers; chordoma;chronic lymphocytic leukemia; chronic myelogenous leukemia; chronicmyeloproliferative disorders; colon cancer; colorectal cancer;craniopharyngioma; cutaneous T-cell lymphoma; endocrine pancreas isletcell tumors; endometrial cancer; ependymoblastoma; ependymoma;esophageal cancer; esthesioneuroblastoma; Ewing sarcoma; extracranialgerm cell tumor; extragonadal germ cell tumor; extrahepatic bile ductcancer; gallbladder cancer; gastric (stomach) cancer; gastrointestinalcarcinoid tumor; gastrointestinal stromal cell tumor; gastrointestinalstromal tumor (GIST); gestational trophoblastic tumor; glioma; hairycell leukemia; head and neck cancer; heart cancer; Hodgkin lymphoma;hypopharyngeal cancer; intraocular melanoma; islet cell tumors; Kaposisarcoma; kidney cancer; Langerhans cell histiocytosis; laryngeal cancer;lip cancer; liver cancer; malignant fibrous histiocytoma bone cancer;medulloblastoma; medulloepithelioma; melanoma; Merkel cell carcinoma;Merkel cell skin carcinoma; mesothelioma; metastatic squamous neckcancer with occult primary; mouth cancer; multiple endocrine neoplasiasyndromes; multiple myeloma; multiple myeloma/plasma cell neoplasm;mycosis fungoides; myelodysplastic syndromes; myeloproliferativeneoplasms; nasal cavity cancer; nasopharyngeal cancer; neuroblastoma;Non-Hodgkin lymphoma; nonmelanoma skin cancer; non-small cell lungcancer; oral cancer; oral cavity cancer; oropharyngeal cancer;osteosarcoma; other brain and spinal cord tumors; ovarian cancer;ovarian epithelial cancer; ovarian germ cell tumor; ovarian lowmalignant potential tumor; pancreatic cancer; papillomatosis; paranasalsinus cancer; parathyroid cancer; pelvic cancer; penile cancer;pharyngeal cancer; pineal parenchymal tumors of intermediatedifferentiation; pineoblastoma; pituitary tumor; plasma cellneoplasm/multiple myeloma; pleuropulmonary blastoma; primary centralnervous system (CNS) lymphoma; primary hepatocellular liver cancer;prostate cancer; rectal cancer; renal cancer; renal cell (kidney)cancer; renal cell cancer; respiratory tract cancer; retinoblastoma;rhabdomyosarcoma; salivary gland cancer; Sezary syndrome; small celllung cancer; small intestine cancer; soft tissue sarcoma; squamous cellcarcinoma; squamous neck cancer; stomach (gastric) cancer;supratentorial primitive neuroectodermal tumors; T-cell lymphoma;testicular cancer; throat cancer; thymic carcinoma; thymoma; thyroidcancer; transitional cell cancer; transitional cell cancer of the renalpelvis and ureter; trophoblastic tumor; ureter cancer; urethral cancer;uterine cancer; uterine sarcoma; vaginal cancer; vulvar cancer;Waldenstrom macroglobulinemia; or Wilm's tumor.

In some embodiments, the brain cancer or tumor is selected from thegroup consisting of brain stem glioma, central nervous system atypicalteratoid/rhabdoid tumor, central nervous system embryonal tumors,astrocytomas, craniopharyngioma, ependymoblastoma, ependymoma,medulloblastoma, medulloepithelioma, pineal parenchymal tumors ofintermediate differentiation, supratentorial primitive neuroectodermaltumors and pineoblastoma.

VIII. Definitions

Technical and scientific terms used herein have the meanings commonlyunderstood by one of ordinary skill in the art to which the presentinvention pertains, unless otherwise defined. Materials, reagents andthe like to which reference is made in the following description andexamples are obtainable from commercial sources, unless otherwise noted.

For convenience, the meaning of certain terms and phrases employed inthe specification, examples, and appended claims are provided below.Other terms and phrases are defined throughout the specification.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise.

The term “about” means that the number comprehended is not limited tothe exact number set forth herein, and is intended to refer to numberssubstantially around the recited number while not departing from thescope of the invention. As used herein, “about” will be understood bypersons of ordinary skill in the art and will vary to some extent on thecontext in which it is used. If there are uses of the term which are notclear to persons of ordinary skill in the art given the context in whichit is used, “about” will mean up to plus or minus 10% of the particularterm.

“Individual,” “subject,” “host,” and “patient,” used interchangeablyherein, refer to any mammalian subject for whom diagnosis, treatment, ortherapy is desired. In one preferred embodiment, the individual,subject, host, or patient is a human. Other subjects may include, butare not limited to, cattle, horses, dogs, cats, guinea pigs, rabbits,rats, primates, and mice.

“Cancer,” “neoplasm,” “tumor,” “malignancy” and “carcinoma,” usedinterchangeably herein, refer to cells or tissues that exhibit anaberrant growth phenotype characterized by a significant loss of controlof cell proliferation. There are several main types of cancer. Carcinomais a cancer that begins in the skin or in tissues that line or coverinternal organs. Sarcoma is a cancer that begins in bone, cartilage,fat, muscle, blood vessels, or other connective or supportive tissue.Leukemia is a cancer that starts in blood-forming tissue, such as thebone marrow, and causes large numbers of abnormal blood cells to beproduced and enter the blood. Lymphoma and multiple myeloma are cancersthat begin in the cells of the immune system. Central nervous systemcancers are cancers that begin in the tissues of the brain and spinalcord. The methods and compositions of this invention particularly applyto precancerous, malignant, pre-metastatic, metastatic, andnon-metastatic cells.

The terms “treatment,” “treating,” “treat,” and the like refer toobtaining a desired pharmacological and/or physiologic effect in a tumorpatient. The effect can be prophylactic in terms of completely orpartially preventing tumor or symptom thereof and/or can be therapeuticin terms of a partial or complete stabilization or cure for tumor and/oradverse effect attributable to the tumor. Treatment covers any treatmentof a tumor in a mammal, particularly a human. A desired effect, inparticular, is tumor response, which can be measured as reduction oftumor mass or inhibition of tumor mass increase. In addition to tumorresponse, an increase of overall survival, progress-free survival, ortime to tumor recurrence or a reduction of adverse effect also can beused clinically as a desired treatment effect.

As used herein, the term “administering” includes directly administeringto another, self-administering, and prescribing or directing theadministration of an agent as disclosed herein.

As used herein, the phrases “effective amount” and “therapeuticallyeffective amount” mean that active agent dosage or plasma concentrationin a subject, respectively, that provides the specific pharmacologicaleffect for which the active agent is administered in a subject in needof such treatment. It is emphasized that an effective amount of anactive agent will not always be effective in treating theconditions/diseases described herein, even though such dosage is deemedto be an effective amount by those of skill in the art.

As used herein, the term “active agent” is any small molecular drug,protein, functional nucleic acid, or polynucleic acid encoding afunctional nucleic acid that is useful for treating a subject. Theactive agent can be any of the anti-neoplastic drugs, functional acids,interferon type I agonists or type II agonists described herein.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in vivowithout excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio.

The term “endocytosis” encompasses (1) phagocytosis and (2) pinocytosis,itself a category inclusive of (2a) macropinocytosis, which does notrequire receptor binding, as well as of (2b) clathrin-mediatedendocytosis, (2c) caveolae-mediated endocytosis and (2d)clathrin-/caveolae-independent endocytosis, all of which tend to accessthe late-endosome/lysosome pathway. The interaction between the ligandon a minicell and a mammalian cell surface receptor, the presentinventors discovered, activates a particular endocytosis pathway,involving receptor mediated endocytosis (rME) to thelate-endosomal/lysosomal compartment. By virtue of such an endocytosispathway, the present inventors further discovered that the minicellswere able to release their payload into the cytoplasm of the targetmammalian cell. In the event the payload is an encoding nucleic acid,the nucleic acid not only is not completely degraded in thelate-endosomal/lysosomal compartment, but also is expressed in thetarget mammalian cell.

The following examples are illustrative only, rather than limiting, andprovide a more complete understanding of the invention. The examplesdemonstrate that drug resistant tumor cells can be effectively treatedin-vivo by (1) administration of targeted recombinant minicells carryingRNAi sequences designed to reduce or eliminate expression of drugresistance encoding gene(s), and (2) administration of targeted,drug-packaged minicells carrying the drug to which the cancer cells aremade sensitive.

The following examples are provided to illustrate the present invention.It should be understood, however, that the invention is not to belimited to the specific conditions or details described in theseexamples. Throughout the specification, any and all references to apublicly available document, including a U.S. patent, are specificallyincorporated by reference.

WORKING EXAMPLES Example 1: Pre-Clinical Studies in Mice

This example showed that minicells (EDVs) provided efficient delivery ofchemotherapy drugs and inhibited tumor growth in mice xenograft models.The EDV targeted technology has been tested on mouse xenograft models ofvarious cancers including colon cancer, breast cancer, ovarian cancer,leukaemia, lung cancer, mesothelioma, and uterine cancer. In addition,various targeting moieties were utilized, as EDVs were targeted to tumorcell surface receptors including EGFR, Human epidermal growth factorreceptor 2 (HER2), Mesothelin (MSLN), and CD33. Finally, the testedtargeted EDVs comprised a wide variety of cytotoxic drugs, includingdoxorubicin, paclitaxel, monastrol, irinotecan, super-cytotoxic drugssuch as PNU-159682, and a novel thymidylate synthase inhibitor(OSI-7904). PNU-159682 is an anthracycline analogue which is thousandsof times more cytotoxic than doxorubicin. OSI-7904 is a benzoquinazolinefolate analog with antineoplastic activity. As a thymidylate synthaseinhibitor, OSI-7904 noncompetitively binds to thymidylate synthase,resulting in inhibition of thymine nucleotide synthesis and DNAreplication.

In all cases, tumor stabilisation or regression was observed when usingspecifically-targeted and drug-packaged EDVs, even with large (>1000mm³) tumors (see Table 5). Control mice treated with free drug (notpackeged in an EDV) showed the expected toxicity of phlebitis andeventually lost weight and died. These toxicities were not observedfollowing repeat administration of EDV-packaged drug.

Surprisingly, even though the concentration of cytotoxic drug deliveredvia EDVs was up to 8,000 times less than systemic delivery, thecytotoxic drug concentration delivered by EDVs was sufficient tomaximise anti-tumor efficacy.

TABLE 5 Summaries of mouse xenograft studies Human cancer Type of EDV,Dose xenograft Results Reference ^(EGFR)EDVs_(Dox/Pac), 1 × 10⁸ Breastcancer Tumor stabilisation or MacDiarmid et al., 2007b; per doseMDA-MB-468 regression FIG. 4A and 4B ^(EGFR)EDVs_(Dox), 1 × 10⁸ Lungcancer Tumor stabilisation or MacDiarmid et al., 2007b; per dose A549regression FIG. 4D ^(CD33)EDVs_(Dox), 5 × 10⁸ Promyelocytic Tumorstabilisation or MacDiarmid et al., 2007b; per dose leukaemia HL-regression FIG. 5A 60 ^(HER2)EDVs_(Dox), 1 × 10⁸ Ovarian cancer Tumorstabilisation or MacDiarmid et al., 2007b; per dose SKOV3 regressionFIG. 5B ^(EGFR)EDVs_(Dox), 1 × 10⁸ Breast cancer Tumor stabilisation orMacDiarmid et al., 2007b; per dose (comparison MDA-MB-468 regressionFIG. 5C with liposomal doxorubicin) ^(EGFR)EDVs_(Dox), dose Breastcancer Tumor stabilisation or MacDiarmid et al., 2007b; escalationMDA-MB-468 regression FIG. 5D ^(EGFR)EDVs_(Dox), 1 × 10⁸ Breast cancerTumor stabilisation or MacDiarmid et al., 2007b; per dose (stabilityMDA-MB-468 regression FIG. 5E comparison of fresh vs reconstituted EDVs)^(EGFR)EDVs_(Mon), 1 × 10⁸ Breast cancer Tumor stabilisation orMacDiarmid et al, 2007a; per dose MDA-MB-468 regression FIG. 1A^(EGFR)EDVs_(OSI-794), 1 × 10⁸ Colon cancer Tumor stabilisation orMacDiarmid et al., 2007a; per dose HT29 regression FIG. 1B^(EGFR)EDVs_(Dox/Pac), 1 × 10⁹ Colon cancer Tumor stabilisation orMacDiarmid et al., 2007a; per dose HCT-116 regression FIG. 4a^(EGFR)EDVs_(Irino), 1 × 10⁹ Colon cancer Tumor growth slowed MacDiarmidet al., 2009; per dose Caco-2 (stabilisation/ FIG. 5a(+/−^(EGFR)EDVs_(shMDR1))* regression with ^(EGFR)EDVs_(shMDR1))^(EGFR)EDVs_(Dox), 1 × 10⁹ Uterine cancer Tumor growth slowed MacDiarmidet al., 2009; per dose MES-SA (stabilisation/ FIG. 6a(+/−^(EGFR)EDVs_(shMDR1))* regression with ^(EGFR)EDVs_(shMDR1))^(EGFR)EDVs_(Dox), 1 × 10⁹ Breast cancer Tumor stabilisation or Tayloret al., MAbs, 7(1): 53-65 per dose MDA-MB-468 regression 2015 (2015);FIG. 7 ^(MSLN)EDVs_(Dox), 1 × 10⁹ Mesothelioma Tumor stabilisation orAlfaleh et al., PLoS One, 12: 1- per dose H226 regression 21 (2017);FIG. 5a

Example 2: Pre-Clinical Studies in Dogs

This example showed that minicells (EDVs) loaded with drugs could besafely administered to dogs.

Canine toxicology studies in dogs with a range of endogenous tumors(n=41) showed that up to 98 doses of targeted and drug-packaged EDVscould be safely administered to a single animal over the course of morethan 2 years. There were mild spikes in temperature (increases of up to1° C.) with concomitant elevation of Interleukin-6 (IL-6),Interleukin-10 (IL-10), and Tumor necrosis factor alpha (TNF-α),following doses in some dogs, however this was not associated with anysignificant adverse events.

Furthermore, pre-clinical studies in dogs showed that CD3-targeted,doxorubicin loaded EDVs can inhibit tumor growth. Two dogs with advancednon-Hodgkin's lymphoma were treated with CD3-targeted,doxorubicin-packaged EDVs, and both demonstrated marked tumor regressionas was evident by highly significant reductions in lymph node size.MacDiarmid et aL, 2007b. Over 60% of dogs with hemangiosarcoma showedtumor stabilisation or regression when treated with CD33-targeted anddoxorubicin-packaged EDVs.

In another study of dogs (n=17) with late-stage brain cancer, theanimals were treated with EGFR-targeted EDVs loaded with doxorubicin.MacDiarmid et al., 2016. Up to 98 repeat doses were administered for asingle dog (with 11 dogs receiving >20 doses) at a concentration of1×10^(10 EGFR)minicells_(Dox) with no signs of toxicity observed. Theobjective response rate was 23.53% (4 of 17 dogs; 95% confidenceinterval, 6.8-49.8%). Of the 15 dogs evaluated for tumor response, 2 hadcomplete responses (CR) to therapy, 2 had partial responses (PR) totherapy (90-98.95% reduction in tumor volume), 10 had stable disease(SD), and 1 showed progressive disease (PD).

Bio-distribution studies using ¹²³Iodine radio-labelled, EGFR-targetedEDVs in two dogs with brain cancer showed localisation of targeted EDVsto brain tumors, suggesting that EDVs can circumvent the blood-brainbarrier to enter the tumor environment. Some localisation in thegastrointestinal track suggests excretion via the faeces.

Example 3: Pre-Clinical Studies in Monkeys

This example showed that minicell (EDV) technology is well tolerated bymonkeys.

Three rhesus monkey trials were performed to assess the toxicity ofempty EDVs (up to 2×10¹⁰ per dose), EGFR-targeted, doxorubicin-loadedEDVs (up to 2×10¹⁰ per dose), and EGFR-targeted, paclitaxel-loaded EDVs(up to 1×10¹¹ per dose). Monkeys were treated with EDVs once weekly for5 weeks (35 day repeat dose testing).

As seen with dogs, there were transient spikes in temperature (increasesof up to 1° C.) with concomitant elevation of IL-6 post-dose. Theinflammatory marker C-reactive protein was also increased at thesetimes, however no significant toxicities or adverse events wereobserved. A mild elevation of TNF-α was seen over the course oftreatment with EGFR-targeted, doxorubicin-loaded EDVs only. A total of72 monkeys have been safely administered with EDV technology.

Example 4: Inflammatory and Immunological Responses in the Pre-ClinicalStudies

This example showed that only minor inflammatory responses were observedin the pre-clinical mice studies (Example 1), the pre-clinical dogstudies (Example 2), and the pre-clinical monkeys study (Example 3).These responses resolved as quickly as 4 hours post-dose. No othersignificant changes in haematological or biochemical parameters wereobserved. Animals remained healthy in appearance and behaviourthroughout the course of treatments.

Formation of anti-product antibodies was evaluated in canine studies andin the monkey trials. The immune responses considered with respect toadministration of targeted EDVs were:

-   -   Serum antibody responses to the EDV-surface exposed        immunodominant antigen being the O-polysaccharide component of        LPS (IgG or IgM responses). Anti-O-polysaccharide antibody        responses are T-cell independent and do not exhibit memory        responses.    -   Serum antibody responses to the mouse IgG monoclonal antibody        used in construction of the BsAb to target the EDVs to tumor        cell surface receptors (e.g., EGFR).

In dogs with hemangiosarcoma or brain cancer, the serum anti-LPS IgGtitres rose to a mean of approximately 10,000 by Dose 3-4 of targetedand doxorubicin-packaged EDVs. Subsequent dosing did not result in anyfurther elevation of titre. Anti-LPS IgG titres in the 3 monkey trials(healthy animals) generally showed a mild rise over the first 2-3 dosesbefore plateauing. The response was largely dose-dependent, rising to amaximum titre of just over 100 for the highest dose levels of^(EGFR)EDV_(Dox). These are considered weak antibody responses sinceanti-O-polysaccharide antibody titres expected in vaccines againstGram-negative bacteria are generally in the millions.

Anti-LPS IgM titre responses in monkey trials were also mild, rising tojust over 100 on treatment with ^(EGFR)EDV_(Dox) and up to 1,000 ontreatment with non-targeted EDVs. Titres were not augmented furtherafter Doses 3-4.

Immunogenic responses to monoclonal antibodies used in construction ofthe BsAbs were also measured in monkey studies, with a mild rise intitre observed in response to the EGFR antibody in monkeys treated withEGFR-targeted EDVs (mouse IgG). These results suggest thatadministration of BsAb-targeted, drug-packaged EDVs may not elicitsignificant anti-LPS immune responses that could prevent theeffectiveness of subsequent doses. This is particularly relevant forcancer patients, whose immune system is likely to be compromised, as itsuggests that repeat dosing is likely to be a viable treatment option.

Example 5: First-in-Man, Phase 1 Clinical Trial EvaluatingErbitux-Targeted, Paclitaxel-Packaged EDVs (^(EGFR(Erb))EDVs_(Pac)) inAdvanced Solid Tumors

This example showed the promising result of using minicells (EDVs) todeliver Paclitaxel (Taxol®) to advanced solid tumors.

In this trial, it was shown that ERBITUX (cetuximab)-Targeted,Paclitaxel-Packaged EDVs are well tolerated in human patients, but asignificant number of the patients had to end the study because ofadverse events or dose-limiting toxicity in a clinical trial.Furthermore, although this treatment strategy achieved stabilization ofthe disease, none of the patients in this study exhibited a partial orcomplete response to the treatment. The results of this trial data arepublished in Solomon et al., 2015.

The First-in-Man trial was designed as a dose escalation study todetermine the safety, tolerability and maximum tolerated dose orrecommended phase 2 dose of EGFR-targeted, paclitaxel-loaded EDVs(^(EGFR(Erb))EDVs_(Pac)). Note that the antibody used for targeting EDVsto EGFR is based on the Erbitux sequence. Other objectives were toassess immune and inflammatory responses to IV administered^(EGFR(Erb))EDVs_(Pac), and to assess response to therapy according toRECIST criteria.

The study was conducted at 3 oncology clinics in Melbourne, Australia,and was registered with the Australian New Zealand Clinical TrialsRegistry (number ACTRN12609000672257). The final study report isavailable and the study has been published in Solomon et al, 2015.

Patients were adults of at least 18 years of age with advancedepithelial malignancies for which standard curative treatment was notavailable.

^(EGFR(Erb))EDVs_(Pac) was administered weekly as a 20-minute IVinfusion in cycles consisting of 5 weeks of treatment. This was followedby a treatment-free week in which patients underwent radiologicalassessment of their tumors with MRI, CT, and or FDG-PET. Patients couldcontinue to receive further cycles of treatment if the tumor remainedstable or was responding to treatment, or if they were deriving clinicalbenefit from the therapy, and they did not experience any dose limitingtoxicities (DLTs) or other adverse events (AEs) requiringdiscontinuation of treatment.

A total of 236 doses were delivered over 7 dose levels: 1×10⁸, 1×10⁹,3×10⁹, 1×10¹⁰, 1.5×10¹⁰, 2×10¹⁰ and 5×10¹⁰ EGFR(Erb) targeted EDVscomprising Paclitaxel per dose. Twenty-two of the 28 patients completedat least 1 full cycle of treatment (5 weekly doses), with one patientreceiving 45 doses over 9 complete cycles (approximately 14 months). Notreatment-related deaths occurred. The maximum tolerated dose wasidentified as 1×10^(10 EGFR(Erb))EDVs_(Pac), with significant toxicitiesobserved above this dose level, particularly in the form of prolongedfever and transient elevation of liver function tests (LFT). Thetreatment was generally well tolerated with acceptable safety findingsin the indicated population.

A summary of the clinical study and findings is presented in Table 6below.

TABLE 6 Summary of clinical data - ^(EGFR(Erb))EDVs_(Pac) Number ofPatients Percent Dose levels 1 × 10⁸ 6/28 21.4% 1 × 10⁹ 6/28 21.4% 3 ×10⁹ 4/28 14.3%  1 × 10¹⁰ 6/28 21.4% 1.5 × 10¹⁰  3/28 10.7%  2 × 10¹⁰1/28 3.6%  5 × 10¹⁰ 2/28 7.1% Length of treatment¹ At least 1 full cycle22/28  78.6% <1 complete cycle 6/28 21.4% Adverse events Alltreatment-related 24/28  85.7% Rigors 16/28  57.1% Pyrexia 13/28  46.4%Serious adverse events 5/28 17.9% Dose limiting toxicities 8/28 28.6%Withdrawal due to AE 4/28 14.3% Response² Stable disease 10/22  45.5%Progressive disease 12/22  55.5% ¹One full cycle consisted of 5 weeklydoses. ²Response was evaluated at completion of 1 full cycle oftreatment.

The most common adverse events that were at least probably related tostudy treatment were low-grade pyrexia (fever) and rigor (chills),experienced in up to 60% of patients (Grade 1-2 severity). Most patientsexperienced mild transient elevations of the cytokines IL-6, IL-8 andIL-10 at 4 hours post-dose. Levels generally returned to baseline within24 hours of receiving the dose. This is consistent with a minorinflammatory response to treatment.

Five patients experienced treatment-related adverse events that wereconsidered serious, and 8 patients experienced dose limiting toxicity oradverse events that that required dose reduction. These events aresummarised in Table 7 and described in the narrative below.

TABLE 7 Serious adverse events or dose limiting toxicities Number ofAdverse events patients Percent Serious DLT Musculoskeletal andconnective tissue disorders Arthritis reactive 1/28 3.6% Y Y Nervoussystem disorders Syncope 1/28 3.6% Y N Metabolism and nutritiondisorders Hypophosphatemia 1/28 3.6% N Y Immune system disordersCytokine release syndrome¹ 1/28 3.6% Y N Investigations Elevated liverfunction tests (ALT, 5/28 17.9% 2* 2* AST) General disorders andadministration site conditions Pyrexia 2/28 7.1% Y N Vascular disordersHypotension 1/28 3.6% Y Y

One patient at the 1×10⁸ dose level experienced elevated LFTs meetingthe DLT criteria. This event was not considered serious, and thedefinition of DLT was amended for subsequent dose levels. Four patientsat dose levels above the MTD experienced elevated LFTs not meeting theamended DLT criteria, however these patients also experienced serioustreatment-associated clinical symptoms (pyrexia, rigors, nausea,vomiting) and the safety committee decided on dose reduction.

Three patients were recruited into the first dose level,1×10^(8 EGFR(Erb))EDVs_(Pac). One patient experienced a grade 3 drop inphosphate levels after 3 of his 5 doses. In each case the levelsreturned to normal by 24 hours post-dose, and there were no clinicalsymptoms. Another patient experienced asymptomatic grade 3 elevations inthe liver enzymes alanine transaminase (ALT) and aspartate transaminase(AST) at 4 hours post-Dose 3, which returned to baseline by the nextdose. These events met the protocol's original definition of doselimiting toxicity (DLT), and the ongoing patients' doses were reduced to5×10^(7 EGFR(Erb))EDVs_(Pac), with one patient receiving a total of 45doses over the study duration. A further 3 patients were recruited toreceive 1×10^(8 EGFR(Erb))EDVs_(Pac), and no drug related adverse eventswere reported by these 3 individuals. The protocol's definition of DLTwas amended to exclude biochemical abnormalities that resolved within 7days of treatment.

The dose was escalated to 1×10^(9 EGFR(Erb))EDVs_(Pac). Two days afterthe second dose one patient experienced severe joint pain. This wasaccompanied by a significant rise in the cytokine interferon-suggestinga viral infection. The patient was admitted for observation and waslater diagnosed with reactive arthritis, which was classed as a seriousadverse event (SAE). After some consideration the safety committeedecided to proceed cautiously and defined this event as a DLT.Therefore, this cohort was also extended to 6 patients. No otherpatients on the trial experienced similar events. At the completion ofthe first cycle the safety data supported escalating the dose further.

A cohort of three patients was recruited to the next dose level,3×10^(9 EGFR(Erb))EDVs_(Pac). One patient was withdrawn after receivingonly 1 dose due to rapidly progressive disease, and subsequently died asa result of disease. A fourth patient was recruited at the same doselevel. The patients all tolerated this dose without any major concernsand the safety data supported escalating the dose further. One patientin this cohort achieved stabilized disease after the first cycle andcompleted two cycles with 10 doses in total. One patient received only 4of 5 doses due to disease infiltration of his bone marrow.

A cohort of three patients was recruited to the next dose level,1×10^(10 EGFR(Erb))EDVs_(Pac). The patients tolerated this dose levelwithout any major concerns and at the completion of cycle 1 the safetydata supported escalating the dose further. Two of the three patientsachieved stabilized disease and completed three and five cycles of 15and 25 doses respectively.

Two patients were recruited to the next dose level,5×10^(10 EGFR(Erb))EDVs_(Pac). They received one dose at this level andboth experienced a grade 3-4 rise in the liver enzymes ALT and AST.These changes were transient and as such did not meet the protocol'samended definition of a DLT, however, as the patients experienced otherAEs such as fever, rigors, and nausea (in one case resulting inhospitalisation for a SAE), the decision was made to reduce the doselevel to 1×10^(10 EGFR(Erb))EDVs_(Pac). These patients also experiencedconsiderable elevation of the inflammatory markers IL-6, IL-8, IL-10 andTNF-α. One of these two patients went on to achieve stabilized diseaseand completed two full cycles following dose reduction.

To identify the maximum tolerate dose (MTD) one patient was recruited tothe intermediate dose level of 2×10^(10 EGFR(Erb))EDVs_(Pac). Thepatient received one dose at this level and similarly experienced grade3-4 transient elevations in ALT and AST, with fever, rigors, nausea andvomiting, and elevation of inflammatory markers. Clinically significantelevations in lactate dehydrogenase (LDH) and gamma glutamyltransferase(GGT) were also observed. Again, though these parameters did not meetthe protocol's amended definition of a DLT, the elevated liver enzymeswere considered to be SAEs, and it was judged clinically appropriate toreduce the dose to 1×10^(10 EGFR(Erb))EDVs_(Pac). This patient achievedstabilized disease and completed four cycles receiving 19 doses.

In a further attempt to identify the MTD, three patients were recruitedto the intermediate dose level of 1.5×10^(10 EGFR(Erb))EDVs_(Pac). Oneof these patients received their first dose without any adverse reactionhowever they did not continue treatment due to rapidly progressivedisease. Another patient experienced grade 3 hypotension, which wasconsidered a dose limiting serious adverse event, and hence their doselevel was reduced to 5×10^(9 EGFR(Erb))EDVs_(Pac). The final patient inthis cohort experienced a grade 3 rise in AST with treatment-associatedsymptoms (fever, rigors, vomiting) after their first dose, as well aselevation of inflammatory markers, and subsequent doses were reduced to1×10^(10 EGFR(Erb))EDVs_(Pac).

It was concluded, therefore, that the MTD for ^(EGFR(Erb))EDVs_(Pac) wasa dose level of 1×10¹⁰ and a further 3 patients were recruited to thisdose level. One patient was withdrawn from the study after one dose dueto presumed cytokine release syndrome. The patient had a pre-existingcough and experienced occasional episodes of syncope due to asupraclavicular mass pressing on the brachiocephalic veins. Between 2-4hours post-dose the patient became febrile, began coughing, andexperienced 3-4 episodes of syncope witnessed by staff. The patient wasadmitted for observation and diagnosed with cytokine release syndrome,although no increase in IFN-γ was detected. The patient experiencedelevation of IL-6, IL-8 and IL-10, similar to that observed in otherpatients at or above this dose level, which likely represents aninflammatory response to the bacterial component of the drug productrather than typical cytokine release syndrome. While this incident didrepresent an SAE, the safety committee decided this did not meet the DLTcriteria due to pre-existing disease involvement. The remaining twopatients completed the first cycle of treatment without any majorconcerns.

No deaths resulted from treatment-emergent adverse events. Overall thetreatment was well tolerated and there are no particular safety concernsfor the intended target population.

Antibodies to Salmonella typhimurium (anti-LPS) and Erbitux at screeningwere negative in all patients. All patients, with the exception of one,developed positive Salmonella antibody titres following treatment withthe ^(EGFR(Erb))EDVs_(Pac) (27/28=96%). Anti-LPS antibody titres reacheda peak by Dose 3 (Day 15) and were maintained at that level despiterepeat dosing. No patients developed positive Erbitux antibody titres.

The 22 patients who completed cycle 1 were evaluated for tumor response.The best response observed was stable disease (SD) (no patients achieveda partial or complete response according to RECIST criteria). Stabledisease was observed in 10/22 (45.5%) of patients at the end of cycle 1,with 12/22 (55.5%) demonstrating progressive disease (PD). One patientin dose level 1 completed nine full treatment cycles, her disease beingalternately stable and progressive from the end of Cycle 4. Hers was thelongest time to development of PD, 197 days.

In conclusion, the first in man study demonstrated that EDVs packagedwith paclitaxel are well tolerated and 45.5% of the patients exhibitdisease stabilization. This example also demonstrates that it isdesirable to improve cancer treatment strategies to improve survival anddisease response.

Example 6: Phase 1 Clinical Trial Evaluating EGFR-Targeted,Doxorubicin-Packaged EDVs (^(EGFR(V))EDVs_(Dox)) in RecurrentGlioblastoma

This is example showed that treatment with EGFR-Targeted,Doxorubicin-Packaged minicells (EDVs) was well tolerated in patientssuffering from Recurrent Glioblastoma. 50% of the patients exhibitedstabilization of the disease, but no patients experienced partial orcomplete response. Whittle et al., J. Clin. Neurosci., 22(12): 1889-1894(2015).

The Recurrent glioblastoma trial was designed as a dose escalation studyto determine the safety, tolerability and maximum tolerated dose orrecommended phase 2 dose of EGFR-targeted, doxorubicin-loaded EDVs(^(V)EDVs_(Dox)). Note that the antibody used for targeting EDVs to EGFRis the same as the antibody for the current protocol (Vectibix-basedsequence). Other objectives were to assess immune and inflammatoryresponses to IV administered ^(V)EDVs_(Dox), and to assess response totherapy according to Response Assessment in Neuro-Oncology (RANO)criteria.

The study began on 5 Feb. 2013 and was concluded on 26 Jun. 2014. It wasconducted at 4 oncology clinics in Sydney and Melbourne, Australia, andwas registered with the Australian New Zealand Clinical Trials Registry(number ACTRN12613000297729). A final clinical study report isavailable. The study has been published based on draft listings inWhittle et al., J. Clin. Neurosci., 22(12): 1889-1894 (2015).

Patients were adults of at least 18 years of age with pathologicallydocumented and definitively diagnosed recurrent World HealthOrganization (WHO) Grade IV glioblastoma, who had experienced diseaserecurrence or progression following receipt of standard of care therapy(including maximum safe surgical resection, standard adjuvantradiation/temozolomide, and maintenance temozolomide treatment).

^(V)EDVs_(Dox) was administered weekly as a 20-minute IV infusion incycles consisting of 8 weeks of treatment. At the end of each cycle,patients underwent radiological assessment of their tumors with magneticresonance imaging (MRI). Patients could continue to receive furthercycles of treatment if the tumor remained stable or was responding totreatment, or if they were deriving clinical benefit from the therapy,and they did not experience any DLTs or other AEs requiringdiscontinuation of treatment.

A total of 197 doses were delivered over 3 dose levels: 2×10⁹, 5×10⁹ and8×10^(9 V)EDVs_(Dox) per dose. Eight of the 14 patients completed atleast 1 full cycle of treatment (8 weekly doses), with one patientreceiving 47 doses over almost 6 complete cycles (approximately 12months). No treatment-related deaths occurred, and no patientsexperienced a dose limiting toxicity or other adverse events requiringdiscontinuation of treatment. A summary of the clinical study andfindings is presented in Table 8 below.

TABLE 8 Summary of clinical data - ^(EGFR(V))EDVs_(Dox) Number ofPatients Percent Dose levels 2 × 10⁹ 3/14 21.4% 5 × 10⁹ 3/14 21.4% 8 ×10⁹ 8/14 57.1% Length of treatment¹ At least 1 lull cycle 8/14 57.1% <1complete cycle 6/14 42.9% Adverse events All treatment-related 13/14 92.9% Pyrexia 7/14 50.0% Nausea 6/14 42.9% Rigor 6/14 42.9% Seriousadverse events 2/14 14.3% Dose limiting toxicities 0/14 0.0% Withdrawalsdue to AEs 0/14 0.0% Best response² Stable disease 3/6  50.0%Progressive disease 3/6  50.0% Survival³  >5 months 8/8  100.0% ≤5months 0/8  0.0% Percentage survival⁴ 3/8  37.5% ¹One full cycleconsisted of 8 weekly doses. ²For patients who completed at least 1cycle of treatment. ³Median historical survival for recurrentglioblastoma is approximately 5 months. ⁴Number and percentage ofpatients alive at 2 years.

The most common adverse events that were at least probably related tothe study treatment were low-grade pyrexia (fever), nausea, and rigor(chills), experienced in up to 50% of patients (generally Grade 1-2severity). Most patients experienced mild transient elevations of thecytokines IL-6, IL-8, IL-10, and TNF-α at 3 hours post-dose. Levelsgenerally returned to baseline within 24 hours of receiving the dose.This is consistent with a minor inflammatory response to treatment.

Five patients experienced treatment-related AEs of Grade 3 or higherseverity according to the National Cancer Institute's Common TerminologyCriteria for Adverse Events (NCI-CTCAE). These events are summarised inTable 9 below and described in the narrative below.

TABLE 9 Grade 3 or higher treatment-related adverse events (AE) to^(V)EDVs_(Dox) Number of patients Adverse events (N = 14) PercentSerious Total patients with ≥Grade 3 severity 5 35.7 AEs Blood andlymphatic system disorders 1 7.1 Lymphopenia 1 7.1 Metabolism andnutrition disorders 2 14.3 Hypophosphatemia 2 14.3 Investigations 1 7.1Alanine aminotransferase 1 7.1 increased Aspartate aminotransferase 17.1 increased Gamma-glutamyltransferase 1 7.1 increased Musculoskeletaland connective tissue 1 7.1 disorders Generalised muscle 1 7.1 Yesweakness Vascular disorders 1 7.1 Hypotension 1 7.1 Yes

Two patients experienced adverse events of ≥Grade 3 severity that wereconsidered serious. One patient at the 5×10⁹ dose level was hospitalisedin the evening following Dose 2 of Cycle 1 for a serious adverse eventof Grade 3 body weakness accompanied by Grade 1 fever. This was notconsidered a dose limiting toxicity as the patient was positive foranti-product antibodies (antibodies to the Salmonella LPS component ofthe EDV) at study enrollment. This patient went on to receive 2 furtherdoses of treatment with no repetition of the event.

Another patient at the 8×10⁹ dose level experienced a serious adverseevent of Grade 3 symptomatic hypotension 4 hours post-Dose 1 of Cycle 1,which resulted in hospitalisation for IV hydration. This was notconsidered a DLT as it was likely attributable to an E. Coli urinarytract infection, for which the patient was treated. The patient received3 subsequent additional doses of study drug.

Three patients experienced an adverse event of ≥Grade 3 severity thatwere not considered serious. One patient experienced Grade 3 increasesin liver enzymes after some doses. These were not considered doselimiting toxicity or serious adverse events as they were asymptomaticand transient, returning to baseline in between doses. Two patientsexperienced Grade 3 hypophosphatemia, and in one instance this wasaccompanied by Grade 4 lymphopenia. These were also not considered doselimiting toxicity or serious adverse events as they were transient anddid not require therapeutic intervention.

Overall the treatment was well tolerated and there are no particularsafety concerns for the intended target population.

To evaluate immune response to the EDV treatment, antibodies toSalmonella were assessed at screening. Thirteen of 14 patients (93%)were negative for Salmonella antibodies at screening. One patientassigned to dose Level 2 was positive at screening. All patients showedan initial rise in antibody titre through to dose 3. The titres weremaintained with no further augmentation over subsequent doses, despiteone patient receiving a total of 47 doses. No patient developedantibodies to Vectibix.

To evaluate efficacy, eight patients who completed Cycle 1 wereevaluated for tumor response. No patient experienced complete or partialremission, however 50% demonstrated stable disease and no patientexperience disease progression. One patient was reported to have stabledisease for the whole duration of study, receiving almost 6 full cyclesof treatment (approximately 12 months).

The eight patients who completed at least 1 full cycle of treatment werefollowed for survival. All 8 patients survived beyond the medianhistorical survival of 5-7 months, with a median OS of 15.1 months(range 9.1 to >18.4). Four subjects were alive at last contact and werecensored at this time. Two of these (14.3%) who completed at least 1cycle of treatment survived for >18 months.

In conclusion, the treatment for recurrent glioblastoma withEGFR-Targeted, Doxorubicin-Packaged EDVs showed promising results withfew adverse events and 50% of the patients experienced stabilization ofthe disease. However, there is a need for improved treatment strategies.

Example 7: Phase 1 Clinical Trial Evaluating EGFR-Targeted EDVs Packagedwith a MicroRNA-16 Mimic (^(EGFR(V))EDVs_(miRNA16a)) in Mesothelioma

This example showed that EGFR-Targeted EDVs Packaged with a MicroRNA-16gave partial response in one mesothelioma patient out of 16 patientstested for efficacy; whereas 62.5% of the patients experiencedstabilized disease, and 31.3% of the patients experienced progressivedisease. The trial data are published in van Zandwijk et al., LancetOncol., 18(10): 1386-1396 (2017) and Kao et al., Am. J. Respir. Crit.Care Med., 191(12): 1467-1469 (2015). This treatment strategy wasgenerally well tolerated.

^(V)EDVs_(miRNA16a) was evaluated in an open-label, multi-centre,exploratory Phase 1 study in subjects with recurrent malignant pleuralmesothelioma (MPM). The primary end points of the trial were toestablish the maximum tolerated dose and DLTs of ^(V)EDVs_(miRNA16a), toevaluate the effect of multiple dosing, and to detect early signs ofefficacy with ^(V)EDVs_(miRNA16a). Note that the antibody used fortargeting EDVs to EGFR was the same as the antibody for the currentprotocol (Vectibix-based sequence). Secondary endpoints of the trialwere to assess the quality of life in patients receiving^(V)EDVs_(miRNA16a) and to monitor changes in Eastern CooperativeOncology Group (ECOG) performance status and pulmonary functionparameters during treatment. Exploratory endpoints evaluated changes inimmune and cytokine markers during treatment.

To be eligible, patients must have had histological or cytologicaldocumentation of MPM with evidence of EGFR expression in their tumortissue. Patients included men and women aged 18 years or older with anECOG performance status of 0 or 1 and a life expectancy of at least 3months. Patients must have displayed disease progression during orfollowing the administration of standard 1^(st) or 2^(nd) line therapyregimens and were required have adequate bone marrow, liver and renalfunction.

^(V)EDVs_(miRNA16a) was administered weekly or twice weekly as a20-minute IV infusion in cycles consisting of 8 weeks of treatment. Atthe end of each cycle, patients underwent radiological assessment oftheir tumors. Tumor response was assessed according to the modifiedresponse evaluation criteria in solid tumors (RECIST) criteria.Spirometry, FDG-PET scan and CT scans were used to assess diseaseextent.

The trial was initiated on 2 Oct. 2014 at three oncology clinics inSydney, Australia, and was concluded on 24 Nov. 2016. The study wasregistered with the Australian New Zealand Clinical Trials Registry(number ACTRN 12614001248651) and on ClinicalTrials.gov (numberNCT02369198). A total of 27 patients were recruited over 5 cohorts, with26 patients receiving a total of 316 doses of ^(V)EDVs_(miRNA16a) (onesubject died before receiving any treatment and was excluded fromfurther analysis). This study has been published in Kao et al, 2015 andvan Zandwijk et al, 2017.

The dose levels evaluated were 5×10⁹ once or twice weekly, and 2.5×10⁹twice weekly. To avoid increased cytokine reactions, dose adaptationwhereby the dose was gradually increased from 1×10⁹ to the Phase 1equivalent dose was also evaluated. A dexamethasone (dex) adaptationwhereby dex was gradually decreased for pre-medication of subsequentdoses was also evaluated. The MTD was identified as5×10^(9 V)EDVs_(miR16a) once weekly. The treatment was generally welltolerated with acceptable safety findings in the indicated population.

A summary of the clinical study and findings is presented in Table 10below.

TABLE 10 Summary of clinical data - ^(EGFR(V))EDVs_(miRNA16a) # ofPatients¹ Percent Dose levels 5 × 10⁹ weekly 6/26 23.1% 5 × 10⁹ twiceweekly 4/26 15.4% 5 × 10⁹ weekly + dose 6/26 23.1% escalation 2.5 × 10⁹twice weekly + 2/26 7.7% dose escalation 5 × 10⁹ weekly + dose 8/2630.8% escalation + dex adaptation Length of treatment² At least 1 fullcycle 16/26  61.5% <1 complete cycle 10/26  38.5% Adverse events Alltreatment-related 26/26  100.0% Infusion-related reactions⁴ 25/26  50.0%Non-cardiac chest pain 14/26  42.9% (tumor pain) Serious adverse events8/26 30.8% Dose limiting toxicities 3/26 11.5% Withdrawals due to AEs2/26 7.7% Best response³ Partial response 1/16 6.3% Stable disease10/16  62.5% Progressive disease 5/16 31.3% ¹One of 27 patients enrolleddied before receiving treatment and was excluded from further analyses.²One full cycle consisted of 8 weeks of treatment (8 weekly doses or 16bi-weekly doses). ³For patients who completed at least 1 cycle oftreatment. ⁴Infusion-related reactions included any of the following:chills, rigors, pyrexia, tachycardia, night sweats, or hypertension.

16 of the 24 patients completed at least 1 full cycle of treatment, with2 patients receiving at least 40 doses in total (≥5 full cycles oftreatment). The best response observed was a partial response in 1patient. This patient demonstrated a near complete remission in responseto treatment with ^(V)EDVs_(miR16a), as described below. Ten patients(62.5%) demonstrated stabilized disease and 5 patients (31.3%)demonstrated progressive disease. The median survival was 36.5 weeks, or8.4 months (range 9.3→119.6 weeks), with 9 patients (60.0%) survivingfor ≥6 months and 5 of these alive and well at >12 months from the startof treatment. See FIG. 4.

Of particular note, one patient (patient #5 from Cohort 1) displayed adramatic clinical response at the end of the first cycle (see Kao etal., Am. J. Respir. Crit. Care Med., 191(12): 1467-1469 (2015)). At theend of the 8-week period, a “complete” metabolic response was evident onhis PET-CT scan, and a partial response was noted on the chest CT scanand confirmed 4 weeks later. The objective imaging response wasaccompanied by a marked improvement in respiratory function testparameters.

The most common treatment-related adverse events were infusion-relatedreactions (96.2%) which included chills, rigors, pyrexia, tachycardia,or hypertension (night sweats were also included in this category). Themajority of these were mild to moderate in severity. Non-cardiac chestpain at the tumor site was also experienced by 14 patients (53.8%) afterinfusion. These reactions were addressed by an amendment to the protocolin which all subjects received an adapted escalating dose schedule inCycle 1, resulting in fewer infusion-related reactions and of lesserseverity. Laboratory examination revealed a transitory rise ininflammatory cytokines and neutrophils, and a transient decrease inlymphocytes shortly after ^(V)EDVs_(miRNA16a) infusion in the majorityof patients, consistent with a mild inflammatory response.

8 patients experienced 9 serious adverse events that were at leastpossibly related to treatment. Non-cardiac chest pain andinfusion-related reactions both occurred in 2 patients. Three patientsexperienced dose limiting toxicities and an additional 2 subjectsexperienced toxicities that were considered dose-limiting but did notfit the criteria for a dose limiting toxicity as they occurred outsideof the dose limiting toxicity window. No treatment-related deathsoccurred. These events are summarized in Table 11 below and described inthe narrative below.

TABLE 11 Treatment-related SAEs or dose limiting toxicities Dose Numberof reduction/ Adverse events patients Percent DLT withdrawal Generaldisorders Non-cardiac 2/26 7.7%   Y (1)   Y (1) chest pain(tumor-related) Infusion-related 2/26 7.7%   Y (1)   Y (1) reactionDizziness, 1/26 3.8% N N confusion, cold sweat Cardiac events Ischaemia(with 1/26 3.8% N Y ECG changes) Cardiomyopathy 1/26 3.8% Y Y(post-infusion reaction) Other events Dysphagia 1/26 3.8% N NAnaphylactoid 1/26 3.8% N Y reaction

Three patients were recruited into Cohort 1 (5×10⁹ weekly). One of theseexperienced a dose limiting toxicity of non-cardiac chest pain aroundthe tumor site. This subject went on to a reduced dose, with subsequentescalation back to full strength. This cohort was expanded to enroll atotal of 6 subjects, with no further DLTs.

Two of 2 subjects in Cohort 2 (5×10⁹ twice weekly) experiencedtoxicities leading to dose reduction or study withdrawal. The first wasan infusion-related reaction which was classified as a dose limitingtoxicity. This subject went on to a reduced dose, with subsequentescalation back to full strength. The second event was persistent ECGchanges with concurrent coronary ischaemia, which was not classified asa dose limiting toxicity as it occurred in the 4^(th) week of dosing(discussed further below). This subject was removed from study and nofurther subjects were enrolled to this cohort (maximum administereddose).

Six subjects were enrolled to an additional cohort (Cohort 3, 5×10⁹weekly+dose escalation) where all subjects began on a reduced dose withsubsequent escalation to full strength to minimise infusion reactions tostudy medication. No subjects experienced a dose limiting toxicity inthis cohort.

Two subjects were enrolled to Cohort 4 (2.5×10⁹ twice weekly+doseescalation) and no dose limiting toxicities were experienced. Howeverdue to the substantial clinical burden associated with twice weeklydosing it was decided to discontinue recruitment to this cohort.

Nine subjects were enrolled to an additional cohort (Cohort 5, 5×10⁹weekly+dose escalation+dex adaptation) to evaluate a dosing regimenwhich included dose escalation and also gradual reduction ofdexamethasone premedication. See FIG. 4. One subject did not receive anytreatment in this cohort. Of the 8 remaining subjects, 2 experiencedtoxicities leading to dose reduction or study withdrawal. The first wasTakotsubo (stress-related) cardiomyopathy, which was classified as adose limiting toxicity (discussed further below). This subject waswithdrawn from study. The second was an anaphylactoid reaction, whichwas not classified as a dose limiting toxicity as it occurred in the7^(th) week of dosing. This subject was, however, removed from thestudy, and recruitment to this cohort was halted. The maximum tolerateddose was determined to be 5×10^(9 V)EDVs_(miRNA16a) once weekly.

A number of cardiac events were noted in this study which had notpreviously been observed in trials of different EDV products for otherindications. Two subjects experienced serious adverse events of cardiacevents resulting in dose reduction or withdrawal (ischaemia andTakotsubo cardiomyopathy). The ischaemia was considered unlikely to bedue to study treatment, as the event occurred 7 days post-dose and thesubject had a previous history of coronary artery disease. This subjectexperienced ECG changes during earlier doses. The cardiomyopathy eventwas preceded by an infusion reaction, possibly as a consequence of dextapering. This subject also had a history of coronary artery disease.Three additional subjects experienced transient ECG changes (T-waveabnormalities) post-dose, which were not classified as serious. Thesewere not associated with elevated troponin, ischaemia or LVEF changes.All events resolved and subjects went on to receive additional doseswith no further ECG abnormalities. Nevertheless, in view of the advancedage of patients with malignant pleural mesothelioma, and the morbiditiesassociated with the disease, these observations collectively led thesafety committee to recommend more stringent cardiac exclusion criteriaand additional cardiac monitoring for the remainder of the study and forany future trials.

Overall treatment with ^(V)EDVs_(miRNA16a) was generally well toleratedup to the maximum tolerated dose of 5×10⁹ EDVs, with no significantconcerns in this particular patient population given adequate monitoringand an adapted escalating dose schedule.

Thus, this example showed promising results in treating mesothelioma byusing EDVs targeting EGFR to deliver miRNA16a to the cancer cells.However, the example results also demonstrate a need for improvedtreatment strategies for mesothelioma.

Example 8: In Vitro Cytotoxicity Assays Revealed that the SupertoxicDrug PNU-159682 Inhibits Proliferation of Tumor Cell Lines to a GreaterExtent than Other Chemotherapy Agents

This example showed that PNU-159682, which is a supertoxic cytotoxicchemotherapy agent, was a more potent inhibitor of cancer cell growththan a wide range of other chemotherapy drugs.

PNU-159682 is a highly potent metabolite of the anthracyclinenemorubicin (MMDX) and is more than 3,000-times more cytotoxic than itsparent compound (MMDX and doxorubicin); thus, PNU-159682 is considered a“supertoxic” chemotherapy drug. Use of such drugs is generally notpossible with typical chemotherapy treatments as the toxicity levels ofsupertoxic drugs result in severe adverse events, including death.

PNU-159682 and the other indicated chemotherapeutic agents were added totumor cell lines at the concentrations indicated in FIGS. 5A-5B-10A-10B.

All cells were incubated for a further 72 hrs followed by thecolorimetric MTS cell proliferation assay (Cory et al., Cancer Commun.,3(7): 207-12 (1991)) using the CellTiter 96 AQu_(eous) One Solution CellProliferation Assay (Promega Corp., Madison, Wis., USA), according tothe manufacturer's instructions. The colorimetric measurements were readat 490 nm.

Greater Cytotoxicity:

The results of these in vitro cytotoxicity assays showed that PNU-159682exhibited greater cytotoxicity against the human lung cancer cell lineA549 as compared to the cytotoxicity observed by a range of knownchemotherapeutic agents as shown in FIG. 5A. In particular, PNU-159682inhibited A549 cells to a much greater degree than doxorubicin. FIG. 5B.

Effectiveness against cancer cells known to exhibit resistance toconventional chemotherapy agents: Further, FIGS. 6A-6B shows thatPNU-159682 and duocarmycin exhibited potent cytotoxic effects againsttwo Adreno-cortical cancer cell lines derived from Stage IV patientsthat were highly resistant to doxorubicin, mitotane, paclitaxel,oxaliplatin, and mitoxantrone. Moreover, FIG. 7 shows that PNU-159682inhibited proliferation of the human breast cancer cell line MDA-MB-468that was resistant to doxorubicin, paclitaxel, and docetaxel. FIGS.8A-8B shows that PNU-159682 inhibited proliferation of the humancolorectal cancer cell lines Caco-2 (FIG. 8A) and HCT116 (FIG. 8B) thatwere resistant to doxorubicin, cisplatin. FIG. 9 shows that PNU-159682could inhibit proliferation the Glioblastoma cell line U87-MG that wasresistant to doxorubicin and paclitaxel IVAX. FIG. 10A shows thatPNU-159682 could inhibit proliferation of human pancreatic cell linesthat were gemicitabine sensitive (MiaPaca-2 cells, FIG. 10A) even thoughthese cells were resistant to doxorubicin, gemzar, mathotrxate,oxaliplatin, irinotecan, and 5-Fluoro Uracil (5-FU). FIG. 10B shows thatPNU-159682 could inhibit proliferation of human pancreatic cell linesthat were gemicitabine-resistant cells (MiaPaca-2 GemR cells, FIG. 10B)even though these cells were resistant to gemzar.

These data demonstrate that it would be highly desirable to usesupertoxic chemotherapy agents such as PNU-159682 in cancer therapies asthe drug can be useful in treating cancers that demonstrate resistanceto conventional, non-supertoxic chemotherapy drugs.

Example 9: PNU-159682 Delivered with EGFR Targeted EDVs can OvercomeDrug Resistance in Human Lung Cancer Cells in a Mouse Xenograft Model

This example showed that using minicells (EDVs) to deliver a supertoxicchemotherapy agent, such as PNU-159682, effectively inhibits tumorgrowth in a lung cancer xenograft model.

A549 (lung cancer) cells were made doxorubicin-resistant by continuousculture in the presence of doxorubicin (dox) and selecting dox-resistantclones. These cells were then implanted as xenografts in Balb/c nu/numice. When the tumor volumes reached ˜150 mm³, 4 different groups ofmice (n=7 per group) were treated intravenously (IV) with (i) saline,(ii) epidermal growth factor receptor (EGFR) targeting EDVs loaded withdoxorubicin (^(EGFR)EDVs™_(Dox)), (iii) EGFR targeting EDVs loaded withPNU-159682 (^(EGFR)EDVs™₆₈₂), and (iv) non targeted EDVs loaded withPNU-159682 (EDVs™₆₈₂) at the time points indicated with solid arrows inFIG. 11. The composition of an EDV targeting EGFR and loaded withPNU-159682 is depicted graphically in FIG. 1.

The results depicted in FIG. 11 show that ^(EGFR)EDVs™_(Dox) had noanti-tumor efficacy, and therefore, the tumors exhibited dox resistance.In contrast, mice treated with ^(EGFR)EDVs™₆₈₂ showed complete tumorstabilisation. When the saline treated tumors reached tumor volumes inthe range of 500 mm³ to 700 mm³, the treatment was changed to^(EGFR)EDVs™₆₈₂ at the time points indicated with asterisks (*) in FIG.11. Surprisingly, the results showed a highly significant anti-tumorefficacy, even in tumors having reached a volume in the range of 500 mm³to 700 mm³.

Example 10: Delivering Functional DNA Agents with EGFR Targeted EDVsEffectively Inhibits Mesothelioma (MSTO) and Adreno-Cortical Cancer(ACC) Cancer Cell Growth

This example showed that using minicells (EDVs) to deliver siRNAtargeting Polo like kinase 1 (Plk1), siRNA targeting ribonucleotide ereductase enzyme 1 (RRM1), or miRNA16a can effectively inhibit growth ofcancer cells.

Polo like kinase 1 (Plk1) and ribonucleotide reductase enzyme 1 (RRM1)were shown to be over-expressed in several non-small cell lung carcinoma(NSCLC) cell lines including A549, A549MDR (dox resistant A549 cellline, over-expressing the multi-drug resistance membrane pump, MDR),H2122, H358 and H441. FIG. 12 shows expression of GAPDH (G), KSP (K),Plk1 (P), and RRM1(R) and the expression is shown relative to the GAPDHexpression in the indicated NSCLC cell lines.

To test if the Plk1 or RRM1 are useful targets for cancer treatment,inhibiting non-coding small interfering RNAs (siRNAs) targeting RRM1(siRRM1) and Plk1 (siPlk1) were synthesized and packaged in EDVs fordelivery to cancer cell lines.

siRNA targeting RRM1 was found to inhibit proliferation of mesotheliomaand adreno-cortical cancer cells. The EGFR-targeted, siRRM1-packagedEDVs were transfected into MSTO (mesothelioma cell line) or H295R(adreno-cortical cancer cell line). Five days post-transfection, cellproliferation was measured and the results are depicted in FIGS. 13A-13Band showed highly significant inhibition of cell proliferation ascompared to control transfections with non-targeted, siRRM1-packagedEDVs or EGFR-targeted, siNonsense-packaged EDVs.

In a mesothelioma (MSTO) xenograft study in Balb/c nu/nu mice,intravenous (IV) treatment with EGFR-targeted, siRRM1-packaged EDVsshowed highly significant anti-tumor efficacy as compared to saline orEGFR-targeted, siScrambled-packaged EDVs as shown in FIG. 14. The tumorsisolated from mice receiving the siRRM1 packaged EDVs were significantlysmaller than tumors from mice receiving negative controls. FIG. 15.

miRNA16a was found to inhibit proliferation of mesothelioma cancercells. In a mesothelioma (MSTO) xenograft study in Balb/c nu/nu mice,intravenous (IV) treatment with EGFR-targeted, miRNA16a-packaged EDVsshowed highly significant anti-tumor efficacy compared to saline orEGFR-targeted, siScrambled-packaged EDVs as shown in FIG. 14. The tumorsisolated from mice receiving the miRNA16a packaged EDVs weresignificantly smaller than tumors from mice receiving negative controls.FIG. 15.

siPLK loaded EDVs targeted to EGFR (^(EGFR)EDV™_(siPLK)) inducedapoptosis in cancer patient-derived tumor Spheroids (ACC001) as shown inFIGS. 16A-16B. EDVs without any payload (^(EGFR)EDV™), or EDVs with RNAinterference molecules targeting the irrelevant luciferase sequence(^(EGFR)EDV™_(luciferase)) were used as a negative control. Compared tothese negative controls, ^(EGFR)EDV™_(siPLK) induced apoptosis in theACC001 spheroids as determined by measurements of cellular debris (FIG.16A), and measurements of Annexin/propidium iodide (PI) ratio (FIG.16B). ^(EGFR)EDV™_(siPLK) treatment also resulted in a significantnumber of cells in sub-G1 cell cycler arrest as compared to the negativecontrols as shown in FIGS. 17A-17D. Thus, inhibiting Plk1 expressionwith siRNA is an effective strategy for inducing apoptosis and cellcycle arrest in ACC001 adreno cortical cancer cells.

Example 11: Delivering Interferon Type I Agonists with EGFR TargetedEDVs Augments Anti-Tumor Efficacy of EDVs Loaded with Cytotoxic Drugs ina Xenograft Model

This example showed that using minicells (EDVs) to deliver achemotherapy agent combined with an interferon type I agonist, was aneffective strategy for treating a cancer such as lung cancer xenografts.The interferon type I agonist can be in the same or a different minicellas the chemotherapy agent. In the present example, the chemotherapyagent is the supertoxic drug PNU-159682 and the interferon type Iagonist is a 40mer double stranded DNA.

A549 (lung cancer) xenografts in Balb/c nu/nu mice were treated withvarious EDV combinations by intravenous injection in the tail vein asdepicted in FIG. 18. The mice were treated with: (i) solidtriangle=^(EGFR)EDVs_(PNU-59682)+EDVs_(40mer), (ii) solidcircle=^(EGFR)EDVs_(PNU-59682), (iii) opensquare=^(EGFR)EDVs_(PNU-159682)+EDVs, (iv) opentriangle=^(EGFR)EDVs_(PNU-59682)+EDVs_(50mer), and (v) solidsquare=saline. The is a type I interferon agonist.

The mice were treated with these EDVs combinations at day 24, 27, 29,31, 34, 36, and 38 after the xenograft implantation as indicated with uparrows in FIG. 18. As shown in FIG. 18, all combinations of EDVs testedresulted in stabilizing the tumor growth. In contrast, the salinetreated control group exhibited tumor growth up to a volume of 650 mm³On day 36 and 38, the saline group mice with tumor volume of ˜650 mm³were treated with ^(EGFR)EDVs_(PNU-59682)+EDVs_(50mer) as indicated bythe down arrows in FIG. 18.

Treating mice having tumors with a large volume of ˜650 mm³ withminicells (EDVs) comprising PNU-159682 and EGFR targeting(^(EGFR)EDVs_(PNU-59682)) combined with EDVs comprising 40mer doublestranded DNA (EDVs_(40mer)) resulted in a dramatic regression of thetumors. Specifically, in just 5 days the tumor volumes decreased from˜650 mm³ to ˜250 mm³—or a 62% reduction in size in 5 days. The resultsare summarized in the table below.

TABLE 12 Phase II Treatment Phase I Starting at Group Treatment FIG.Results days 36 and 38 Results 1 ^(EGFR)EDVs_(PNU-159682) + FIG. 18,Tumor growth N/A N/A EDVs_(40mer) solid stabilization triangle 2^(EGFR)EDVs_(PNU-159682) FIG. 18, Tumor growth N/A N/A solid circlestabilization 3 ^(EGFR)EDVs_(PNU-159682) + FIG. 18, Tumor growth N/A N/AEDVs (no payload) open square stabilization 4 ^(EGFR)EDVs_(PNU-159682) +FIG. 18, Tumor growth N/A N/A EDVs_(50mer) open stabilization triangle 5Saline FIG. 18, tumor growth Treatment with In 5 days, tumors having asolid square up to a volume ^(EGFR)EDVs_(PNU-159682) + large volume of~650 mm³ of ~650 mm³ EDVs_(40mer) decreased to ~250 mm³ - or a 62%reduction in tumor size in 5 days

Furthermore, EDVs comprising 40mer double stranded DNA (EDVs_(40mer)) incombination with minicells (EDVs) comprising PNU-159682 and EGFRtargeting (^(EGFR)EDVs_(PNU-59682)) induced more significant regressionof tumors as compared to tumor cells treated with^(EGFR)EDVs_(PNU-159682) alone in a mouse xenograft model of lungcancer, as shown in FIG. 19. In FIG. 19, Balb/c nu/nu mice were treatedwith (i) solid circle=^(EGFR)EDVs_(PNU-159682), (ii) solidtriangle=^(EGFR)EDVs_(PNU-159682)+EDVs₄₀mer, or (iii) solidsquare=saline. The results are summarized in the table below.

TABLE 13 Group Treatment FIG. Results 1 ^(EGFR)EDVs_(PNU-159682) FIG.19, solid circle Slight tumor size reduction (from a tumor volume ofabout 275 mm³ to 260 mm³) 2 solid triangle = FIG. 19, solid triangleSignificant tumor reduction, from a tumor ^(EGFR)EDVs_(PNU-159682) +volume about 275 mm³ to about 175 mm³) EDVs_(40mer) 3 Saline FIG. 19,solid square Significant tumor growth.

In conclusion, a type I IFN agonist packaged in a minicell augments theanti-neoplastic effects of ^(EGFR)EDVs_(PNU-159682) treatment.

Example 12: Clinical Evaluation of EDVs_(PNU-59682) with Adjuvant Type IIFN Agonists (EDV_(40mer) or EDVs_(60mer)) and Type II IFN Agonists(Imukin)

This example showed that type I and type II IFN agonists augment theanti-cancer effect of ^(EGFR)EDVs_(PNU-159682) in human patientssuffering from advanced solid tumors.

Remarkably, even in an advanced stage pancreatic cancer patient, thistreatment produced a 90% drop in tumor marker levels after only 3 dosesand the patient exhibited markedly improved life quality.

Treatment with Minicells comprising drug, type I IFN agonist, and typeII IFN agonist: The inventors of the present disclosure performedclinical case studies where subjects received targeted and loaded EDVsin combination with minicells loaded with the type 1 IFN agonists 40merdouble stranded DNA (EDVs_(40mer)) (type 1 IFN agonist) or 60mer doublestranded DNA (EDVs_(60mer)) (type 1 IFN agonist).

Three subjects with advanced solid tumors received combination treatmentof ^(EGFR(V))EDVs_(PNU-59682) with adjuvant EDVs_(40mer) (type 1 IFNagonist) as part of the Designer EDV Study (Melbourne, Australia). Twoadditional patients received loaded and targeted EDVs in combinationwith EDVs_(60mer) (type 1 IFN agonist) under compassionate uselegislation. One compassionate-use patient diagnosed with Stage IVpancreatic cancer received treatment with ^(EGFR(V))EDVs_(PNU-59682) andEDVs_(40mer) (type 1 IFN agonist) or EDVs_(60mer) (type 1 IFN agonist),and a second compassionate use patient diagnosed with recurrentadreno-cortical cancer received ^(EGFR(V))EDVs_(PNU) andEDVs_(60mer)+Imukin (type II IFN).

Clinical Study

Minicells Comprising Drug and Type I IFN Agonist:

^(EGFR(V))EDVs_(PNU) with adjuvant EDVs_(40mer) (type 1 IFN agonist) wasevaluated in 3 patients in an open-label, single centre, exploratoryPhase 1 study in subjects with advanced solid tumors (Designer EDVStudy). To be eligible, patients were to have histological orcytological documentation of advanced solid tumors with evidence of EGFRexpression in their tumor tissue to facilitate targeting. Patients musthave displayed disease progression during or following theadministration of standard 1st, 2nd or 3rd line therapy regimens. Theprimary end points of the trial were to establish the safety andtolerability of ^(EGFR(V))EDVs_(PNU-159682) with adjuvant EDVs_(40mer)(type 1 IFN agonist).

This trial was initiated at a single centre in Melbourne, Australia, andwas registered with the Australian New Zealand Clinical Trials Registry(number ACTRN 12617000037303).

The 3 patients in the trial received a total of 13 doses of^(EGFR(V))EDVs_(PNU-159682) at 2.5×10⁹ with EDVs_(40mer) (type 1 IFNagonist) at 5×10⁸. Treatment was administered weekly as a 20-minute IVinfusion in cycles consisting of 8 weeks of treatment. At the end ofeach cycle, patients were to undergo radiological assessment of theirtumors.

Available safety data are limited, but the treatment has generally beenwell tolerated with no unexpected adverse reactions to the IP. As seenwith administration of other EDV products, patients generallyexperienced a transient increase in the inflammatory cytokines IL-6,IL-8 and TNF-α, which returned to baseline between treatments doses.Observed changes in haematology parameters largely mirrored changes seenin previous trials with EDV therapeutics, including mild self-limitingelevation of white blood cells (WBC), elevation of neutrophils 3 hourspost-dose, and a concomitant decrease in lymphocytes and monocytes.Parameters returned to normal at the following time point, prior to thenext dose. Some subjects experienced a mild reduction in serum phosphatelevels, which did not require intervention and returned to baselinebetween doses.

Two patients experienced related adverse events involvinginfusion-related reactions, with rigors and fever beginningapproximately 1 hour post-dose. These patients were admitted overnightfor observation and the events resolved by the following day. Onepatient was withdrawn from study due to dose limiting toxicity. Thesecond patient continued the study and received additional doses.

Compassionate Use Studies

The first compassionate use case study took place at Royal North ShoreHospital, Sydney. The patient was a 68 year old female who was diagnosedwith Stage IV pancreatic cancer. She had Whipple surgery(pancreaticoduodenectomy), with gemcitabine as first line treatment. Shealso received the FOLFIRINOX treatment regime but developed metastaticliver disease. Her tumor cells were tested in vitro and found to besensitive to PNU-159682.

The patient received bi-weekly doses of EDV products includingPNU-loaded and EGFR-targeted EDVs in combination with differentimmunomodulatory adjuvants. These were delivered IV as a 20 mL infusion.She received both EGFR(V)-targeted EDVs and ITG(609)-targeted EDVscomprising PNU, and also EDV_(40mer) (type 1 IFN agonist) orEDVs_(60mer) (type 1 IFN agonist) as intended for use in the currentprotocol. In total, the patient received 45 doses of^(EGFR(V))EDVs_(PNU-159682)+EDVs_(40mer) (or the related productEDVs_(60mer)). Doses of ^(EGFR(V))EDVs_(PNU-59682) and^(ITG(609))EDVs_(PNU-159682) were escalated up to a maximum of 2×10⁹ and4×10⁹ respectively, and EDV_(40mer/60mer) were given at a set dose of5×10⁸.

The patient tolerated the treatment very well, with no IP-relatedserious adverse events. Preliminary results indicate a transientincrease in the inflammatory cytokines IL-6, IL-8 and TNF-α post-dose,similar to that seen with administration of other EDV products.

These responses were generally reduced over subsequent doses. Theanti-inflammatory cytokine IL-10 also was transiently increased,post-dose. Interestingly, IFN-α was increased as well at various timepoints throughout the study, which is likely a consequence ofstimulation with EDV_(40mer/60mer). No elevation of IFN-γ was detectedat 2 hours post-dose.

Remarkably, the levels of the patient's tumor marker (CA 19-9) droppedby more than 90% after the first 3 doses, equivalent to only 10 days oftreatment. After 10 doses this had dropped even further, with an almost95% reduction in tumor marker levels. She also demonstrated significantweight gain, in contrast to the cachexic state experienced by mostpatients presenting with stage IV pancreatic cancer, and reported amarked improvement in quality of life. The preliminary safety andefficacy results of this case study are thus extremely promising,particularly given the poor prognosis associated with advancedpancreatic cancer.

Minicells Comprising Drug, Type I IFN Agonist, and Type II Agonist:

The second compassionate use case study in an end-stage adreno-corticalcancer patient with a very heavy tumor burden was treated at Royal NorthShore Hospital (Sydney.

The patient received 10 bi-weekly doses of ^(EGFR(V))EDVsP_(NU)-15₉68₂(minicell comprising an antineoplastic agent, with doses of theantineoplastic agent ranging from 1×10⁹ to 4×10⁹ [EDVs], EDVs_(60mer)(minicell comprising type I IFN agonist, with doses of the type 1 IFNagonist ranging from 5×10⁸ to 2×10⁹ [EDVs]), and Imukin (type II IFN,with doses of the type II IFN ranging from 5 μg (1×10⁵ IU) to 30 μg(6×10⁵ IU)).

The patient tolerated the treatment very well, experiencing only mildelevation of temperature up to 60 minutes post-dose. This is to beexpected on administration of EDV products. Unfortunately, the patienthad a very high disease burden including high levels of cortisol whichis known to be a serious immune system suppressor and the CT scansduring week 7 showed progressive disease and hence the patient was takenoff the study.

In summary, 5 patients received a total of 69 doses of^(EGFR(V))EDVs_(PNU/Dox) or ^(EGFR(V))EDVs_(PNU/PNU)+EDV_(40mer60mer),(type I IFN agonist)±Imukin (type II IFN). The treatments were welltolerated, and addition of immunomodulatory adjuvants did not seem tochange the safety profile of single agent loaded and targeted EDVs.

Example 13: Addition of IFN-γ (Type II IFN Agonist) Augments theAnti-Tumor Efficacy of Epidermal Growth Factor Receptor Targeted EDVsLoaded with Doxorubicin and Cause Tumor Regression in Xenograft Modelsof Various Cancers

This example showed that using minicells (EDVs) to deliver doxorubicincombined with IFN-γ provides improved anti-tumor effects in micexenograft models.

Lung Cancer:

To study the anti-tumor effects of combining ^(EGFR)EDVs_(Dox) and IFN-γ(type II IFN) in a lung cancer model, A549 (lung cancer) xenografts inBalb/c nu/nu mice were established and divided into four groupsreceiving different treatment combinations by intravenous tail veininjection. Group 1 received sterile physiological saline (FIG. 20, opendiamonds). Group 2 received IFN-γ (0.5×10⁴ IU) per dose (FIG. 20, solidtriangles). Group 3 received ^(EGFR)EDVs_(Dox) (FIG. 20, solid squares).Group 4 received ^(EGFR)EDVs_(Dox) and IFN-γ (0.5×10⁴ IU) per dose (FIG.20, solid circles).

Mice treated with ^(EGFR)EDVs_(Dox) achieved tumor stabilisation of A549lung cancer xenografts (FIG. 20, solid squares). In contrast, micetreated with ^(EGFR)EDVs_(Dox) and IFN-γ showed highly significant tumorregression by day 43 after a total of 6 doses (FIG. 20, solid circles).Mice treated with IFN-γ alone showed no anti-tumor efficacy (FIG. 20,solid triangles), and the tumors grew as in the saline treated group(FIG. 20, open diamonds).

Thus, combining ^(EGFR)EDVs_(Dox) and IFN-γ (type II IFN) resulted intumor regression in a mouse xenograft model of lung cancer, assummarized in Table 14, below.

TABLE 14 Group Treatment FIG. Results Group 1 sterile physiologicalsaline FIG. 20, open diamonds no anti-tumor efficacy, and tumors grewGroup 2 IFN-γ (0.5 × 10⁴ IU) per FIG. 20, solid triangles no anti-tumorefficacy, and tumors dose grew Group 3 ^(EGFR)EDVs_(Dox) FIG. 20, solidsquares tumor stabilisation Group 4 ^(EGFR)EDVs_(Dox) and IFN-γ FIG. 20,solid circles highly significant tumor regression (0.5 × 10⁴ IU) perdose by day 43 after a total of 6 doses

Breast Cancer:

To study the anti-tumor effects of combining ^(EGFR)EDVs_(Dox) and IFN-γin a breast cancer model, MDA-MB 468 xenografts in Balb/c nu/nu micewere established and divided into four groups receiving differenttreatment combinations by intravenous tail vein injection. Group 1received sterile physiological saline (FIG. 21, open diamonds). Group 2received IFN-γ (0.5×10⁴ IU) per dose (FIG. 21, solid triangles). Group 3received ^(EGFR)EDVs_(Dox) (FIG. 21, solid squares). Group 4 received^(EGFR)EDVs_(Dox) and IFN-γ (0.5×10⁴ IU) per dose (FIG. 21, solidcircles).

Mice treated with ^(EGFR)EDVs_(Dox) achieved tumor stabilisation ofMDA-MB 468 breast cancer xenografts, but by −day 25 the tumors began togrow again, likely due to development of resistance to doxorubicin (FIG.21, solid squares). In contrast, mice treated with ^(EGFR)EDVs_(Dox) andIFN-γ showed highly significant tumor regression, and by day 30, after atotal of 6 doses, these tumors were more like scar tissue (FIG. 21,solid circles). Mice treated with IFN-γ alone showed no anti-tumorefficacy (FIG. 21, solid triangles), and the tumors grew as in thesaline treated group (FIG. 21, open diamonds). In an additionalexperiment depicted in FIG. 22, mice treated with ^(EGFR)EDVs_(Dox)again achieved tumor regression of MDA-MB 468 breast cancer xenografts,but by −day 23 the tumors began to grow again, likely due to developmentof resistance to doxorubicin (FIG. 22, solid squares).

In contrast, mice treated with ^(EGFR)EDVs_(Dox) and IFN-γ showed highlysignificant tumor regression and by day 28, after a total of 3 doses,these tumors were more like scar tissue (FIG. 22, solid circles). Micetreated with IFN-γ alone showed no anti-tumor efficacy (FIG. 22, solidtriangles), and the tumors grew, as in the saline treated group (FIG.22, open diamonds). Thus, combining ^(EGFR)EDVs_(Dox) and IFN-γ (type IIIFN) resulted in tumor regression in a mouse xenograft model of breastcancer, as summarized in Table 15, below.

TABLE 15 Group Treatment FIG. Results Group 1 sterile physiological FIG.21, open diamonds Exps. #1 and #2: no anti-tumor saline FIG. 22, opendiamonds efficacy, and tumors grew Group 2 IFN-γ (0.5 × 104 IU) FIG. 21,solid triangles Exps. #1 and #2: no anti-tumor per dose FIG. 22, solidtriangles efficacy, and tumors grew Group 3 ^(EGFR)EDVs_(Dox) FIG. 21,solid squares Exp. #1: tumor stabilisation, but by ~day 25 the tumorsbegan to grow again, likely due to development of resistance todoxorubicin FIG. 22, solid squares Exp. #2: tumor regression, but by~day 23 the tumors began to grow again, likely due to development ofresistance to doxorubicin Group 4 ^(EGFR)EDVs_(Dox) and FIG. 21, solidcircles Exp #1: highly significant tumor IFN-γ (0.5 × 104 IU)regression, and by day 30, after a total per dose of 6 doses, thesetumors were more like scar tissue FIG. 22, solid circles Exp. #2: highlysignificant tumor regression and by day 28, after a total of 3 doses,these tumors were more like scar tissue

Lung cancer: To study the anti-tumor effects of combining^(EGFR)EDVs_(Dox) and IFN-γ (type II IFN) in a doxorubicin-resistantlung cancer model, the A549 lung cancer cell line was initially grown inculture in the presence of doxorubicin (Dox), and a Dox-resistantderivative cell line was established. Then, Dox-resistant A549xenografts in Balb/c nu/nu mice were established and divided into fourgroups receiving different treatment combinations by intravenous tailvein injection. Group 1 received sterile physiological saline two timesper week (FIG. 23, open diamonds). Group 2 received ^(EGFR)EDVs_(Dox)(FIG. 23, solid triangles). Group 3 received ^(EGFR)EDVs_(Dox) and IFN-γ(0.75×10⁴ IU) per dose (FIG. 23, solid squares). Group 4 received^(EGFR)EDVs_(Dox) and IFN-γ (0.5×10⁴ IU) per dose (FIG. 23, solidcircles). As depicted in FIG. 23, group 1, 2, and 3 were the indicateddosages two times per week (indicated by solid triangles on the x axisin FIG. 23); whereas group 4 mice were administered the indicated dosagethree times per week (depicted by open triangles on the x-axis in FIG.23). Mice treated twice or three times a week with ^(EGFR)EDVs_(Dox) andIFN-γ (0.5 or 0.75×10⁴ IU) achieved tumor stabilisation of resistantA549 lung cancer xenografts (FIG. 23). In contrast, mice treated with^(EGFR)EDVs_(Dox) showed no anti-tumor efficacy and the tumors grew asin the saline treated group. This result suggests that the inclusion ofIFN-γ (0.5 or 0.75×10⁴ IU) along with ^(EGFR)EDVs_(Dox) in the treatmentof tumors normally resistant to the latter alone is essential to achievetumor stabilisation. Thus, combining ^(EGFR)EDVs_(Dox) and IFN-γ canovercome drug resistance in a lung cancer xenograft model as summarizedin Table 16, below.

TABLE 16 Group Treatment FIG. Results Group 1 sterile physiologicalsaline FIG. 23, open diamonds no anti-tumor efficacy, and 2x per weektumors grew Group 2 ^(EGFR)EDVs_(Dox) FIG. 23, solid triangles noanti-tumor efficacy, and tumors grew Group 3 ^(EGFR)EDVs_(Dox) and IFN-γFIG. 23, solid squares tumor stabilisation (0.75 × 10⁴ IU) per doseGroup 4 ^(EGFR)EDVs_(Dox) and IFN-γ FIG. 23, solid circles tumorstabilisation (0.5 × 10⁴ IU) per dose

Example 14: Treatment of Dogs with Late-Stage Endogenous Tumors with^(EGFR)EDVs_(PNU) or ^(ITG)EDVs_(PNU-159682)+EDVs_(40mer) (Type 1 IFNAgonist)+Imukin (Type II IFN)

This example showed that delivering the supertoxic drug PNU-159682 and atype I IFN agonist (40mer double stranded DNA) with minicell technologyand additionally interferon gamma (Imukin) (type II IFN) was welltolerated in a dog study.

A toxicology study was carried out in dogs with endogenous late-stagecancers. Dogs were companion animals presenting with late stage tumors.Informed consent was obtained from each pet owner.

Thirteen dogs with brain cancer, sarcoma, or melanoma were treated withPNU-loaded EDVs targeted to EGFR or ITG (integrin), in combination withimmunomodulatory adjuvants (EDVs_(60mer/50mer) and/or Imukin). Dogs withbrain cancer or sarcoma were treated with EGFR-targeted EDVs (n=9), anddogs with melanoma were treated with ITG-targeted EDVs (n=4). A total of185 weekly or bi-weekly doses were administered in differentcombinations with or without adjuvants, with up to 73 doses received bya single dog. Doses of EDVs administered were up to 5×10⁹ PNU-loaded andtargeted EDVs, up to 2×10⁹ EDVs_(40mer/50mer), and Imukin at 25 μg/m²per dose. All combinations were generally well-tolerated, with the mostcommon adverse events being similar to those seen on administration ofother EDV products (mild lethargy, fever, nausea, vomiting). Addition ofimmunomodulatory adjuvants did not appear to change the general spectrumor increase the frequency of AEs seen with EDV dosing.

Notable effects on haematological parameters included mild transitoryreduction of leukocyte subsets at 3 hours post-dose (WBC, neutrophils,lymphocytes, monocytes and eosinophils). Similar changes were seen withor without the addition of immunomodulatory adjuvants. It is of interestto note that in other canine and human clinical studies, neutrophilsgenerally increased rather than decreased at 3 hours post-dose. Thismild reduction in neutrophils appears to be specific to treatment ofdogs with PNU-loaded EDVs.

Dogs experienced transient elevation of IL-6, IL-8, IL-10, IL-12p40 andTNF-α at 3 hours post-dose. In general, doses comprisingimmunomodulatory adjuvants resulted in a slightly higher induction ofthese cytokines than doses comprising PNU-loaded EDVs alone. They alsoresulted in reduced levels of IL-2 post-dose. These data provide supportfor the safety and tolerability of immunomodulatory EDV adjuvants usedin combination with anti-neoplastic-loaded and targeted EDVs.

The best response observed was stabilized disease in 6 of 7 evaluableanimals (85.7%), although 1 dog achieved a near partial response (29.8%reduction in tumor size). One dog demonstrated stabilized disease overthe course of the study, receiving 73 doses over more than 11 months oftreatment. This dog exhibited loss of vision due to the tumor masspressing on the optic nerve; however, vision was restored over thecourse of treatment, demonstrating improvement in clinical symptoms.

Example 15: Targeted and Loaded EDVs do not Activate IFN-γ in HumanClinical Studies

This example showed that EDVs packaged with Paclitaxel or Doxorubicininduced a cytokine response in human patients consistent with toll likereceptor activation, but this EDV treatment did not induce an interferontype II response.

To gain insight into the pathways activated by administration oftargeted and loaded EDVs, a panel of cytokines was evaluated in theFirst-in-Man (Example 5 above) and recurrent glioblastoma human clinicaltrials (Example 6 above).

FIGS. 24A-25K shows the cytokine response to ^(EGFR(Erb))EDVs_(Pac)treatment in the First in Man study (Example 5), and FIGS. 25A-25K showsthe cytokine response to ^(EGFR(V))EDVs_(Dox) in the recurrentglioblastoma study. As shown in FIGS. 24A-24K and 25A-25K, IL-6, IL-8,IL-10, and to a lesser extent TNF-α were transiently elevated at 3-4hours post-dose, and returned to baseline by 24 hours or prior to thenext dose for both treatments. The cytokine response to EDVs loaded withPaclitaxel or Doxorubicin is consistent with activation of the toll-likereceptor pathway, and in particular TLR4 which is known to be stimulatedby bacterial LPS. IL-12 was randomly elevated in 2 patients in theFirst-in-Man study only (FIGS. 24A-24K), though elevations were notconsistent with dosing.

Interestingly the type I interferon IFN-α was elevated at random stagesthroughout the study in 3/22 patients in the First-in-Man study (seeFIGS. 24A-24K), and in 1/14 patients in the recurrent glioblastoma study(see FIGS. 25A-25K). Induction of IFN-α was not consistent with dosing.Interferon pathways are generally activated by viral rather thanbacterial stimuli like the TLR pathways, so it is possible that theseselected patients had concurrent unreported viral infections during thestudy (e.g. a cold). However all other cytokines tested, includingIFN-γ, were not affected by dosing with targeted and loaded EDVs. Thissuggests that activation of type II IFN-γ (via addition of Imukin) maybe a viable approach for enhancing the efficacy of targeted and loadedEDVs by stimulating different anti-tumor pathways.

Example 16: Cyto-Immuno-Therapy for Cancer: A Novel Pathway Elicited byTumor-Targeted, Supertoxic Drug-Packaged Bacterially-Derived Nanocells

This example demonstrates that the EDV nanocell targeted to a tumor cellsurface receptor functions as a cancer immunotherapy capable of a dualassault on the tumor by delivering the super cytotoxin PNU-159682 (682)in conjunction with activation of the innate and adaptive immunesystems.

This example shows targeted EDV nanocells delivering 682 activated M1macrophages and NK cells which are capable of killing tumor cells withinthe microenvironment accompanied with a predominantly Th1 cytokineresponse. This is followed by dendritic cell maturation resulting inantigen presentation and generation of tumor specific CD8⁺ T-cells. Thecombination of super cytotoxin delivery and activation of both innateand adaptive antitumor immune responses, results in a potent cancercyto-immunotherapeutic which has potential in clinical oncology.

This example shows for the first time on the novel cyto-immunotherapyfunction of the EDV nanocell, where it is not only capable of deliveringa cytotoxic drug within tumor cells but at the same time of eliciting aninnate and adaptive immune response specifically targeting the tumor.Clinically, in a compassionate use case patient, 682 loaded EDV areshown to overcome drug resistance while stimulating adaptive immunity.Pre-clinical studies demonstrated that the immunotherapeutic pathwayresulting from EDV treatment encompasses an approach which addresses allthe necessary steps needed to mount an effective antitumor response bythe immune system. This example illustrates the ability of the EDV toactivate cells of the innate immune system, including macrophages, NKcells and dendritic cells. This is followed by dendritic cell maturationand antigen presentation leading to an adaptive T-cell response in whichtumor specific cytotoxic T-cells are produced and results in furtherrecruitment of additional immune cells to the tumor microenvironment.This approach circumvents some of the current pitfalls withimmunotherapies by creating an immunogenic tumor microenvironment andalso acting on multiple immune cell subsets thereby avoiding primaryand/or adaptive resistances that may arise in patients.

Results: M1 Macrophage Polarization and Dendritic Cell Maturation inResponse to EDV Treatment:

Due to the fact that the EDV is derived from Salmonella Typhimuriumbacteria, the outer EDV membrane contains a substantiallipopolysaccharide (LPS) content (MacDiarmid et al., 2007b). Theinteraction of high doses of LPS with macrophages is well known toresult in macrophage activation and M1 polarization. To determine if theEDV was capable of eliciting a similar phenotypic response, RAW264.7cells were incubated with Ep-EDV-682 and Ep-EDV and examined for changesin macrophage phenotype and cytokine production. Expression of theco-stimulatory CD86 expression is known to be a phenotypic indicator ofmacrophage polarization as well as a hallmark of the antitumor M1 tumorassociated macrophages (TAMs) (Dong et al., 2016).

Both Ep-EDV-682 and Ep-EDV were capable of eliciting significantincreases in the level of CD86 expression, most likely in response tothe presence of LPS on the EDV surface, whereas 682 alone did not inducethe same response (FIG. 27A). Furthermore, EDV treated RAW cellsdisplayed a significant increase in the pro-inflammatory cytokines TNF-αand IL-6, which have been identified as being responsible for Th1macrophage polarization (Yuan et al., 2015) (FIGS. 35A and 35B).Interestingly, mouse tumor cells (4T1 and CT26Ep12.1) which had beentreated with Ep-EDV-682 followed by co-culture with RAW264.7 cells werealso able to generate a significant increase in CD86 expression on theRAW264.7 cells (FIG. 27B) as well as a significant increase in theproduction of the pro-inflammatory cytokines TNF-α and IL-6. (FIG.27C-27D). However, Ep-EDV or 682 treatment alone was unable to produceany significant change in CD86 expression, and 682 treatments showed noincrease in the production of Th1 cytokines, indicating that cell deathin response to EDV loaded 682 was necessary to fully induce subsequentM1 macrophage polarization.

The effect of tumor cell death in response to Ep-EDV, Ep-EDV-682, and682 treatment on dendritic cell maturation was also examined (FIGS.27E-27I). Bone marrow derived dendritic cells (BMDC) were co-incubatedwith treated tumor cells (4T1 and CT26Ep12.1) for 48 h, followed byassessment of the production of the type 1 interferons IFNα and IFNβ.Increases in type 1 interferon production by dendritic cells is wellestablished as a mechanism of dendritic cell maturation and enhancedantigen presentation as well as being vital for their interaction withother immune cell subsets including NK cells and T-cells(Fitzgerald-Bocarsly and Feng, 2007; Simmons et al., 2012). BMDCco-culture with Ep-EDV-682 treated 4T1 cells showed significantincreases in both type 1 interferons with ˜100 fold increase in IFNαmRNA production and ˜70 fold increase in IFNβ mRNA production (FIG.27E). Similarly, BMDC co-culture with Ep-EDV-682 treated CT26Ep12.1showed ˜300 fold increase in IFNα mRNA production and ˜60 fold increasein IFNβ mRNA production (FIG. 27F).

In addition, to assess if differences in the drug loaded into the EDVhad any effect on type 1 interferon production, CT26Ep12.1 were treatedwith Ep-EDV-Dox and showed a significant ˜20 fold increase in IFNβ mRNAproduction, but only a slight, non-significant increase in IFNα mRNAproduction. Ep-EDV and 682 treatment were unable to elicit increases intype 1 interferon mRNA production in co-cultures with either cell line.However, doxorubicin (Dox) treatment of CT26Ep12.1 did in fact show asignificant ˜5 fold increase in IFNβ mRNA production (FIG. 27F).Doxorubicin treatment of tumor cells is known to result in immunogeniccell death, and is therefore capable of prompting dendritic cellmaturation. However, drug doses well above the IC50 are generallynecessary for this type of death to occur with the drug alone (Showalteret al., 2017). Co-incubation of BMDC with EDV and drug treated 4T1 andCt26Ep12.1 cells resulted in upregulation of CD86, MHC Class II, andCD80 within 24 h (FIGS. 27G-27I) with similar results seen in mouse JAWSII cells (FIGS. 35C-35E). BMDC co-cultured with EDV treated tumor cellsalso exhibited a profound and significant increase in the production ofTNFα, IL-12p40, and IL-6 (FIGS. 27J-27L) Combined, these resultsindicate that EDVs loaded with 682 are capable of polarizing macrophagestowards the M1 antitumor phenotype, as well as promoting dendritic cellmaturation, while 682 alone is unable to elicit a similar response.

Example 17: Effective Delivery of the Super Cytotoxin PNU-159682Generates Tumoricidal CD11b⁺ Innate Immune Cells

Since 682 is a super cytotoxin with IC50s for even drug-resistant cancercells in the pM range (Quintieri et al., 2005), it is unable to be usedclinically due to the severe systemic toxicity associated with suchcompounds (Staudacher and Brown, 2017). However, when encapsulated inthe EDV, super cytotoxins such as 682 can be effectively delivered tothe tumor with few side effects as evidenced by minimal weight loss(≤5%), little to no fur ruffling, and no appearance of lethargy orhunched postures in Ep-EDV-682 treated mice which correlates withprevious studies involving mice treated with EDVs carrying a variety oftherapeutic payloads (MacDiarmid et al., 2009; MacDiarmid et al., 2007a;MacDiarmid et al., 2007b; Sagnella et al., 2018) (FIGS. 35A-35D).

Here, significant tumor regression was seen in Ep-EDV-682 treated BALB/cmice bearing either 4T1 tumors in the mammary fat pad or subcutaneousCT26Ep12.1 tumors (FIGS. 28A-28B). Ep-EDV-682 was also capable ofprompting significant tumor reduction in athymic BALB/c nude micebearing T84 human xenografts and drug resistant A549/MDR xenografts aswell as elicit significant tumor reduction in large (˜600 mm³) A549/MDRtumors (FIGS. 28C-28D).

While it has been well established that the EDV can effectively deliverchemotherapeutics to tumors, initial in vitro experiments indicated thatthe EDV can also behave as an immunotherapeutic in a number of waysincluding, but not limited to, driving M1 macrophage polarization. Toestablish if these results extended to in vivo stimulation of the innateimmune system within the tumor microenvironment, CD11b⁺ immune cellswere isolated from either 4T1 or CT26Ep12.1 mouse tumors which had beentreated with saline, Ep-EDV, or Ep-EDV-682 and co-cultured ex vivo withtheir corresponding tumor cells in an xCELLigence real time cellanalyzer (RTCA) at a 5:1 (Effector:Target) ratio (FIGS. 28E-28G). CD11b⁺cells co-cultured with adherent 4T1 cells, showed an initial adhesionand settling phase resulting in an increase in the cell index followedby an active phase in which cell index decreased steadily if the CD11b⁺cells were effective in killing the adherent tumor cells or increased iftumor cells were not effectively lysed and continued to grow.

As demonstrated, CD11b⁺ cells isolated from the tumors of mice which hadbeen treated with Ep-EDV-682 were highly effective at killing 4T1 cells,while those isolated from either Ep-EDV or saline treated tumors did notkill 4T1 tumor cells (FIG. 28E). Phenotyping of macrophages within the4T1 tumors (CD45⁺ CD11b⁺ Ly6G-Ly6C⁺) showed an increase in the M1/M2ratio as evidenced by an increase in the ratio of CD86:CD206 expressingmacrophages (FIG. 28F). Moreover, CD11b⁺ from Ep-EDV-682 4T1 tumorbearing mice exhibited a 2-fold increase over saline and Ep-EDV treatedmice in the production of macrophage inflammatory protein 1a(CCL3/MIP-1α), when co-cultured ex vivo with 4T1 cells (FIG. 361).Localized production of MIP-1α has been implicated as a major factorresponsible for attracting immune effector cell infiltrates into thetumor microenvironment and potentiating an effector cell mediatedantitumor immune response (Allen et al., 2018; Zibert et al., 2004).

CD11b⁺ cells co-cultured with adherent CT26Ep12.1 cells similarlyexhibited enhanced cytolytic activity of CD11b⁺ cells isolated from thetumors of mice treated with Ep-EDV-682 (FIG. 28G). Overall, CD11b⁺ cellsfrom CT26Ep12.1 tumors were more active than those from 4T1 tumors asindicated by the steady decrease in cell index on the xCELLigence RTCAfor all three treatment groups. However, cytolysis was more pronouncedand began within 1 hr post CD11b⁺ cell addition in the Ep-EDV-682treated group falling to 42% viability at 10h post CD11b⁺ cell addition.In contrast, it took nearly 5h for cytolysis to begin in the Ep-EDVtreated samples and 7h for the saline treated group and viability at 10hpost CD11b⁺ cell addition was 76% and 86% respectively. Flow Cytometricanalysis of the CD11b⁺ cells isolated from the CT26Ep12.1 tumors showeda significant increase in the CD86:CD206 ratio (M1/M2 ratio) in theEp-EDV-682 treated tumors (FIG. 28H).

As with the immunocompetent mouse strains, a shift in macrophagepolarization was also seen in a drug resistant human lung cancerxenograft (A549/MDR) in athymic nude mice where ˜2-3 fold increase inM1/M2 ratio in the spleens of EGFR-EDV-682 treated mice was observed ascompared to saline treated mice or mice treated with EDVs that wereineffective at reducing tumor growth (FIG. 35E). Additionally, a small,but significant increase in M1/M2 ratio was also detected in theEGFR-EDV-682 treated T84 tumors (FIG. 35F).

Similar to both immunocompetent mouse cancer models, CD11b⁺ cellsisolated from the tumors of nude mice bearing A549/MDR tumors treatedwith EGFR-EDV-682 exhibited superior tumor cell cytolysis with 28%cytolysis 6.5h post CD11b⁺ cell addition to A549/MDR tumor cells asdetermined by xCELLigence RTCA compared to those treated with saline(FIGS. 35G and 35H).

Example 18: NK Cells Adopt an Antitumor Phenotype In Vivo FollowingEp-EDV-682 Treatment

The purpose of this example was to determine the impact on NK cellfunction of bacterial minicells comprising an antineoplastic agent.

To explore the effect of EDVs carrying 682 on NK cell function, NK cellswere isolated from spleens of EDV treated and control BALB/c micebearing either 4T1 or CT26Ep12.1 tumors following 2 weeks treatment withEp-EDV-682, Ep-EDV or saline. Splenic NK cells were co-cultured in thexCELLigence RTCA with their corresponding tumor cells at an E:T ratio of20:1 and tumor cell death was analyzed over a 3-4 day period (FIGS.29A-29D).

NK cells of Ep-EDV-682 treated mice in both tumor models demonstratedantitumor properties via significant and potent cytolysis of the targettumor cells, while those treated with saline or Ep-EDV showed little tono cytolytic potential towards their target tumor cells. NK cells frommice bearing CT26Ep12.1 displayed rapid cytolysis of target CT26Ep12.1cells, dropping to nearly 60% target cell viability within the first fewhours of co-culture and maintaining only 18% target cell viability after50 h. NK cells from Ep-EDV treated CT26Ep12.1 had a low level ofcytolytic capacity, with 70-80% target cell viability after 50h, whileNK cells from saline treated mice showed ˜82% target cell viability inthe same time period (FIG. 29D). NK cells from Ep-EDV-682 treated micebearing 4T1 tumors, while still highly active, displayed a more gradualcytolytic profile dropping to ˜36% target cell viability after 70h,while NK cells from saline or Ep-EDV treated mice maintained ≥90% targetcell viability (FIGS. 29A and 29B). Additionally, NK/4T1 co-cultureswith NK cells isolated from mice treated with Ep-EDV-682 exhibited morethan a 2-fold increase in the production of both TNFα and RANTES ascompared to those from saline treated mice (FIGS. 29F and 29G).

NK cells isolated from the spleens of athymic mice bearing eitherA549/MDR or T84 tumors demonstrated similar cytolytic profiles to theimmunocompetent mouse tumor models (FIGS. 36A and 36C). NK cells ofEGFR-EDV-682 treated mice, when co-cultured with their target tumorcells, resulted in under 40% target cell viability in both the A549/MDRand T84 tumors, while the saline controls for both maintained a targetcell viability ≥70%. Examination of Granzyme B production in the T84/NKcell co-cultures revealed significantly higher levels of Granzyme B inthe co-cultures containing the NK cells from the Ep-EDV-682 treated mice(FIG. 36B).

Examination of NK cells within the 4T1 tumors via flow cytometry showedthat NK cells (CD45+, CD11b+, DX5+) in the Ep-EDV-682 treated tumors hada significant increase the NKG2D expression, an NK activating receptorknown to be important in tumor immunosurveillance (FIG. 29E).Upregulation of NKG2D ligands on the tumor cell surface are sufficientto override NK inhibitory signals thus enabling tumor cell cytolysis(Morvan and Lanier, 2016). Screening of mouse tumor cell lines(including CT26Ep12.1 and 4T1) for the NKG2D binding NK ligands RAE-1,H60a, and MULT-1 showed that both 4T1 and CT26Ep12.1 had the highestlevel of expression of H60a of the 4 cell lines screened and this wasthe ligand with the highest overall expression in these two cell lines.Further, the mouse breast cancer cell lines 4T1 and EMT-6 cells showedthe highest expression of RAE-1 in all cell lines screened, however, atmuch lower levels than H60a, while CT26Ep12.1 had only very low levelsof this ligand. Finally, with the exception of the 4T1 cells, all othercell lines showed no expression of MULT-1 (FIG. 29H).

To further examine the role of these ligands and the NKG2D receptor inthe cytolytic activity of the isolated NK cells, NK cells were isolatedfrom the spleens of Ep-EDV-682 treated mice bearing 4T1 tumors. NK cellswere incubated with antibodies designed to block binding of the NKG2Dreceptor to its particular ligand before co-culture with 4T1 cell in thexCELLigence RTCA system (FIG. 29I). Antibodies to RAE-1 resulted in ˜13%inhibition of NK cytolysis of 4T1 cells, while antibodies to H60aresulted in ˜21% inhibition, and combination of the two antibodiesinhibited ˜25% of the cytolytic ability of the NK cells (FIG. 29J).

Example 19: A Predominantly Th1 Cytokine Response within the TumorMicroenvironment is Exhibited Following Ep-EDV-682 Treatment

The purpose of this example was to explore the cytokine milieu withinthe tumor microenvironment following EDV treatment.

4T1 and CT26Ep12.1 tumors were harvested 24h following the finaltreatment and gently dissociated in a non-enzymatic manner ensuring nolysis of cells so that interstitial tumor cytokine levels could beassessed (FIGS. 30A-30B). Due to the significant differences in tumorsizes, tumors were weighed and measured, and cytokine levels werecalculated per gram of tissue to account for these size differences.Both tumors exhibited a significant increase in TNFα within the tumormicroenvironment in response to Ep-EDV-682 treatment, although thisincrease was more pronounced in the CT26Ep12.1 tumors with >10-foldincrease following Ep-EDV-682 treatment compared to saline treatment.Similarly, Ep-EDV-682 treatment resulted in a significant increase inthe interstitial IFNα concentration in both tumors with ˜2 fold increasein IFNα levels in the 4T1 tumors and a 15 fold increase in theCT26Ep12.1 tumors.

The most prominent change in cytokine level with Ep-EDV-682 treatmentwas seen in the CT26Ep12.1 tumors where a 500 fold increase in IFNγlevels occurred, while a small, but significant 2 fold increase in IFNγlevels occurred in the 4T1 tumors. The IL-113 level as a result ofEp-EDV-682 treatment was significantly decreased in 4T1 tumors butshowed an increase, albeit not significant, in the CT26Ep12.1 tumors.Significant increases in IL-2 (˜4 fold) and IL-4 (˜3 fold) occurred inthe 4T1 tumors, while there was no significant change in IL-6 levelsfollowing Ep-EDV-682 treatment. In the CT26Ep12.1 tumors of Ep-EDV-682treated mice, IL-2 levels significantly increased more than 150 fold,while a significant 3 fold increase in IL-6 levels and a 2 foldnonsignificant increase in IL-4 was observed. MIP-1α (CCL3) and RANTES(CCL5), which have been shown to be major determinants of infiltrationby immune cells such as antigen presenting cells, NK cells, and T-cells,were also examined (Allen et al., 2018). Both the 4T1 and CT26Ep12.1exhibited increases in the level of the two chemokines in the Ep-EDV-682treated tumors with a ˜3 fold significant increase in MIP-1α levels inthe CT26Ep12.1 tumors and RANTES levels in the 4T1 tumors treated withEp-EDV-682. Furthermore, a 2-2.5 fold increase in RANTES and MIP-1αlevels occurred in both the CT-26Ep12.1 and 4T1 Ep-EDV treated tumorsrespectively, although this was not significant. Generally, Ep-EDVtreatment resulted in interstitial tumor cytokine and chemokine levelssimilar to the saline treated group.

In addition to examination of the interstitial tumor cytokines, cytokineproduction (TNFα, IFNγ, IL-113, IL-2, and IL-10) by splenocytes fromtreated animals was assessed. Splenocytes were cultured alone orco-cultured with dispersed tumor cells from their corresponding mouse.Systemic treatment with saline, Ep-EDV, and Ep-EDV-682 had nosignificant effect on cytokine production by splenocytes from either the4T1 or Ct26Ep12.1 tumor model (FIGS. 30C-30G). However, when splenocyteswere cultured with their corresponding treated tumor, this was no longerthe case. TNFα production significantly increased in the co-culturesfrom mice treated with Ep-EDV-682 as compared to splenocytes only aswell as the co-cultures from saline and Ep-EDV treated mice from bothtumor models (FIG. 30C). In the 4T1 model, IL-2 production significantlyincreased in the co-cultures from the Ep-EDV-682 treated mice ascompared to the saline treated mice (FIG. 30D). Moreover, there was asignificant decrease in IL-2 production when the splenocytes from salinetreated mice were co-cultured with their corresponding tumor cells. IL-2production increased significantly in all co-cultures as compared to thesplenocytes alone isolated from mice bearing CT26Ep12.1 tumors. The onlysignificant change in IL-10 production occurred in co-cultures from the4T1 model in which samples from saline treated mice exhibited asignificant increase as compared to Ep-EDV-682 treated micecorresponding to the difference seen in vivo (FIG. 30E). IFNγ productionsignificantly decreased between splenocytes alone and co-culturesisolated from saline treated mice bearing 4T1 tumors and significantlygreater in the Ep-EDV-682 treated splenocyte/tumor cell co-cultures thanthose from saline or Ep-EDV treated 4T1 tumor bearing mice (FIG. 30F).In the CT26Ep12.1 tumor model, IFNγ production decreased in theEp-EDV-682 treated splenocyte/tumor cell co-cultures as compared to thesaline and Ep-EDV co-cultures, however this was only significant forEp-EDV. Finally, IL-10 production significantly increased in the salinetreated splenocyte/tumor cell co-cultures from the CT26Ep12.1 treatedmice as compared splenocytes alone and the EDV treatment groups (FIG.30G).

Example 20: Ep-EDV-682 Treatment Lead to the Production of TumorSpecific CD8+ T-Cells

Initial in vitro experiments indicated that EDV treatment can result indendritic cell maturation either via direct interaction or as a resultof cell death in response to a targeted EDV loaded with an effectivechemotherapeutic. Thus, this experiment aimed to examine if this resultcould translate to DC maturation and antigen presentation in vivoresulting in the production of tumor specific CD8⁺ cytotoxic T-cells.

Following 2 weeks treatment, spleens were removed from 4T1 or CT26Ep12.1tumor bearing mice which had been treated with saline, Ep-EDV, orEp-EDV-682 and the CD8⁺ T-cells were isolated. CD8⁺ T-cells were thenadded to the corresponding tumor cells and examined for their ability tospecifically recognize and kill those cells using the xCELLigence RTCA(FIGS. 31A and 31C). CD8⁺ T-cells isolated from mice bearing 4T1 andtreated with saline or Ep-EDV exhibited no cytotoxicity towards 4T1cells, while CD8⁺ T-cells isolated from the mice treated with Ep-EDV-682induced 50% cytolysis of the target cells after 30h (FIG. 31B).

CD8⁺ T-cells isolated from mice bearing CT26Ep12.1 and treated withEp-EDV-682 were highly effective in killing the target CT26Ep12.1 cells,with 81% cytotoxicity seen 20h after the addition of the effector cellsto the target cells (FIG. 31D). Interestingly, even the Ep-EDV treatmentwas able to elicit the production of tumor specific CD8⁺ T-cells in theCT26Ep12.1 model, with 40% cytotoxicity apparent after 20h, while theCD8⁺ T-cells from the saline treated mice showed no specificity towardsthe CT26Ep12.1 cells. Flow analysis of CD8+ T-cells within the 4T1tumors showed a small, but significant increase in the percentage ofCD8+ T-cells within the tumors (CD45⁺, CD3⁺, CD8⁺) of Ep-EDV-682 treatedmice (FIG. 31E). Additionally, a significant 2 fold decrease was seen inthe percentage of regulatory T-cells (CD45⁺, CD3⁺, CD4⁺, CD25⁺) withinthe tumors of Ep-EDV-682 treated mice (FIG. 31F). T-cell numbers in thetumor draining lymph nodes of 4T1 tumor bearing mice were also examinedvia flow. A significant increase in overall T-cell numbers (CD3+) aswell as a significant increase in both CD4+ and CD8+ T-cells numberswere seen in the lymph nodes of Ep-EDV-682 treated mice as compared toboth the saline and Ep-EDV treated mice (FIG. 31G). A significantincrease in mature dendritic cells in the lymph nodes of Ep-EDV-682treated mice bearing 4T1 tumors was also detected (FIG. 31H).Visualization of the interaction between isolated CD8⁺ T-cells fromEp-EDV-682 treated mice with 4T1 cells shows that these T-cells arecapable of attaching to and expelling perforin (green) into the tumorcell (FIG. 31I).

Example 21: Patient Response to EGFR-EDV-682 in a Case of Stage IVPancreatic Ductal Adenocarcinoma

This experiment pertains to the clinical observation of a compassionatecase usage of EGFR-EDV-682 treatment in a patient (CEB) with stage IVpancreatic ductal adenocarcinoma (PDAC).

Diagnostic evaluation of CEB included computerised axial tomography (CT)of the abdomen (May 2017) which revealed multiple low density liverlesions. The tumours were not avid on positron emission tomography(PET). Standard biochemistry and haematology tests were generallyunremarkable. Serum CA19-9 and C-reactive (CRP) protein were alsoassessed. Low serum levels of CA 19-9, a carbohydrate antigen that isexpressed on some gastrointestinal malignancies, particularly pancreaticcancers, have been shown to be a prognostic indicator of overallsurvival and response to therapy. Similarly, elevated CRP levels havealso been shown to be significantly associated with poor clinicaloutcomes (Szkandera et al., 2014). Even after gemcitabine andFOLFIRINOX, CEB presented with a CA19-9 level of >120,000kU/L, 3,000times higher than normal, and a considerable elevated CRP level of 64mg/L.

PDAC cells obtained from resected tumor tissue from both the head andtail of the pancreas were examined for drug sensitivity. Both the headand tail PDAC cells exhibited low sensitivity with partial to noresponse to first and second line drugs (FIG. 37A). In contrast, bothdisplayed extreme sensitivity to 682, with IC50's in the pM range (FIG.37A). Further, epidermal growth factor receptor (EGFR) was found to beoverexpressed with >200,000 copies per cell by flow cytometry (FIGS.37A-37C) and therefore, an ideal receptor for targeting EDVs loaded with682 for treatment.

CEB tolerated the EDVs carrying 682 and targeted to EGFR (E-EDV-D682)very well and reported a dramatic increase in well-being throughout thecycle. Her ECOG performance status fell from 2 to 0 during that time.There was a transient rise in TNFα and IL-6 at 3 hours post dose whichwas highest post dose 1 and was much lower in subsequent doses, possiblyindicative of a tolerance build up. There were no changes inhaematological parameters, and white cell count (WCC) remained normalthroughout the cycle. Biochemistry results were unremarkable, even after14 doses. Of note was the CA19-9 marker which steadily fellfrom >120,000 kU/1 to 5,310 kU/ml at, and the CRP levels which feel from64 mg/L to 7 mg/L at dose 13 (FIGS. 32A and 32B).

Post dose 12, immunophenotyping of major immune cell subsets fromperipheral blood mononuclear cells (PBMCs) revealed changes withinmultiple cell types that may support a favorable anti-tumor response(Gating strategy—FIG. 38). Total CD14+ monocytes, the precursors formacrophages and dendritic cells, were increased from 15.28% to 31.39% atD12 (105% increase) when compared with the screen dose (D1) (FIG. 32C),including the intermediate (CD14+CD16++) monocyte subset (69% increase;FIG. 32D). The intermediate monocytes demonstrate the highest capacityto present antigen to T cells, with superior antigen-specific inductionof IL-12 and IFN-γ (Ziegler-Heitbrock and Hofer, 2013).

A 28% increase in the myeloid dendritic cells (mDC) and a 60% decreasein the plasmacytoid DC (pDC) were also observed (FIG. 32F). Clec9A+myeloid dendritic cells (mDCs) which are responsible for driving a CD8+effector T response also increased (98% at D12) (FIG. 32E). Thisincrease was in concordance with increases in the totalcytotoxic CD8+ Tcells (60%), including effector CD8+ T cells (50% increase) at D12 (FIG.32G). The effector CD8+ T cell pool are CD8+ T cells that have recentlyinteracted with antigen presented by monocytes, macrophages or dendriticcells and contain tumour and/or EDV antigen-specific T cells. CytotoxicCD8+ T cells expressing the exhausted programmed death-1 (PD-1+)phenotype, indicative of prolonged cell activation and susceptibility toPD-1 ligation by tumour cells expressing PD-L1, were decreased at D12 by17% compared to D1. This process commonly occurs within the tumour andtumour-draining lymph nodes. The results observed in this case studyfollow a similar trend to those observed in the pre-clinical mousemodels.

Discussion:

This data demonstrates the ability of targeted EDVs loaded with thesuper-cytotoxin 682 to not only effectively deliver this drug to thetumor site, but also behave as an immunotherapeutic by stimulatingmultiple immune cell subsets. The ability of Ep-EDV-682 treatment topush immune cell subsets including macrophages, NK cells and CD8⁺T-cells towards an antitumor phenotype capable of effectivelyeliminating tumor cells has been demonstrated. When combined with theeffectiveness of the drug, this results in a dual assault on the tumor.

Following intravenous administration, the EDV extravasates to the tumorvia its leaky vasculature where ≥30% of the administered dose oftargeted EDVs carrying their toxic payload deposit directly into thetumor microenvironment within a 2 hr period (MacDiarmid et al., 2007b).EpCAM targeted EDVs bind to surface expressed EpCAM on the tumor cells(4T1 and CT26Ep12.1 in this case), and are then internalized effectivelydelivering their payload (682) directly within the tumor cells. 682 is ahighly potent super cytotoxin resulting in rapid apoptosis within 24h ofbeing delivered to the tumor cells (FIG. 33A). The apoptotic cells andDAMP signals produced by Ep-EDV-682 treatment can then interact withinnate immune cells such as tumor associated macrophages (TAMs) andstimulate upregulation of CD86 and the production of Th1pro-inflammatory cytokines such as TNFα and IL-6 (FIG. 33B).

Furthermore, the EDV itself can also interact directly with TAMsproducing a similar M1 polarization, albeit this would be expected tooccur at very low levels in the current system. Here, the ability ofEp-EDV-682 treatment to shift the M1:M2 balance within the tumormicroenvironment in 4 different tumor models has been demonstrated.Despite differences in the degree of this shift in the different tumormodels, it was shown that the increase in M1 polarization translated toincreased tumor cell lysis by TAMs isolated from the tumors of micewhich had been treated with Ep-EDV-682. In addition to the phenotypicshifts to M1, TAMs from tumors of Ep-EDV-682 treated mice also secretedan increased amount of MIP-1α (FIG. 33C), a chemokine which has beenestablished to play a role in promoting immune cell recruitment, and inparticular tumor infiltration by NK cells, CD4⁺ T-cells and CD8⁺ T-cells(Allen et al., 2018).

EDV treatment allows for in vivo priming and maturation of DCs withinthe tumor microenvironment in response to dying tumor cells (FIG. 33D).During the maturation process, the DCs migrate to the tumor draininglymph nodes for antigen presentation to T-cells thereby increasingproduction of CD4⁺ T-helper cells and tumor specific CD8⁺ CTL initiatingan adaptive immune response to the tumor (FIG. 33E).

In conjunction with enhancing macrophage and DC antitumor functions, EDVtreatment is capable of eliciting NK cell activation leading toincreased cytotoxicity (FIG. 33F). Upregulation of the NKG2D receptorwas observed on NK cells within the tumors of mice treated withEp-EDV-682, and this receptor was demonstrated to contributesignificantly to the cytolytic ability of NK cells isolated fromEp-EDV-682 treated mice. Moreover, immature, intermediate and maturemouse NK cells express both the CCR1 and CCR5 chemokine receptors thatcan bind the chemokines MIP1α and RANTES, both of which are upregulatedin Ep-EDV-682 treated tumors as well as by macrophages and NK cells fromEp-EDV-682 treated mice (Bernardini et al., 2016).

Chemokines, such as MIP1α and RANTES, are responsible for the furtherrecruitment of helper and effector immune cells including NK cells,macrophages, and T-cells to the tumor microenvironment (FIG. 33G) (Allenet al., 2018; Bernardini et al., 2016; Zibert et al., 2004). Followingthe initial innate immune response due to EDV treatment whichencompasses macrophages, NK cells, and DCs, an adaptive immune responseis mounted in which tumor specific CTLs and T-helper cells are producedand then recruited to the tumor site (FIG. 33H). Tumor specific CTLsthen target and lyse tumor cells further contributing to the overridingantitumor environment which has been created by the other immune cellsubsets in combination with the targeted, drug loaded EDVs. Targeted,drug loaded EDV treatment elicits a mainly Th1 response as evidenced bythe increase of Th1 cytokines (TNFα, IFNα, IFNγ, IL-2, and IL-6) withinthe tumor microenvironment. As previously mentioned, innate immune cellsubsets, when activated, become a primary source of one or more of theseparticular cytokines. T-cells are similarly capable of producing all ofthe aforementioned cytokines (Belardelli and Ferrantini, 2002; Lee andMargolin, 2011). Release of these cytokines by either innate immunecells or T-cells are responsible for co-stimulation, activation, growth,and increased antigen presentation of additional immune cells creating afeedback loop which further enhances the antitumor activity of theimmune system FIG. 33I) (Lee and Margolin, 2011).

Methods and Materials

EnGeneIC Dream Vector (EDV):

EDV were produced and purified from a Salmonella enterica serovarTyphimurium (S. Typhimurium) minCDE-strain as previously described(MacDiarmid et al., 2007b). Drug loading, antibody targeting,lyophilization, and dose preparation have been previously described(MacDiarmid et al., 2007b; Sagnella et al., 2018). EDV preparations weresubject to strict quality control in which EDV size and number wereassessed using dynamic light scattering using a Zetasizer Nano Seriesand NanoSight LM20 (Malvern Instrument). Endotoxin levels were assessedusing an Endosafe portable test system (Charles River). Drug wasextracted from EDV™ preparations and quantified via HPLC as previouslydescribed (MacDiarmid et al., 2007b).

Flow Cytometry:

All flow cytometry was performed on a Beckman Coulter Gallios 6C andanalyzed using Kaluza software (Beckman Coulter).

Cell Culture:

RAW264.7 cells (ATCC) were grown to ˜70% confluence in Dulbecco'sModified Eagle Media (DMEM) (Sigma) containing 10% FCS and passagedusing a cell scraper. Mouse tumor cell lines (4T1 and CT26) were grownin monolayers in RPMI-1640 media (Sigma) containing 10% FCS and passaged2-3 times per week using phosphate buffered saline (PBS)/Trypsin EDTA.All cells were maintained in culture at 37° C. in a humidifiedatmosphere containing 5% CO₂ and routinely screened and found to be freeof mycoplasma. EpCAM expression and receptor number in the mouse celllines were quantified using flow cytometry with APC anti-mouse CD326(Biolegend) using Quantum Simply Cellular anti-Rat IgG microspheres(Bangs Laboratory). As CT26 were shown to be negative for EpCAM, cellswere transfected with a pcDNA3. 1+C DYK containing the mouse EpCAM ORFclone (NM_008532.2) (Genescript) using Lipofectamine 2000 (ThermoFisher). G418 selection was used to obtain pure populations of EpCAMexpressing CT26 clones, and cells were screened as described above forEpCAM expression. Clones were examined for growth rate, drug sensitivityand in vivo tumorgenicity, and one that possessed high EpCAM expressionwith the above 3 parameters being similar to the parental CT26 cell linewas selected for all subsequent studies (CT26Ep12.1).

Bone Marrow Derived DCs (BMDC):

Bone marrow was isolated from the femurs and tibias of Balb/c mice.Following red blood cell lysis and washing, cells were resuspended inAIMV+5% FBS+2-mercaptoethanol+penicillin/streptomycin+20 ng/ml GM-CSF(Miltenyi Biotec) and grown for 7 days.

Treatment of RAW264.7 Cells with EDVs:

RAW264.7 cells were seeded in 6-well plates at 3×10⁵ cells per well andincubated overnight. Media was then replaced with fresh media containingone of the following: 1 μg/mL LPS (Sigma); 100 pmol PNU-159682 (NajingLevena); Ep-EDV-682 (500:1 and 1000:1 EDV: cells), Ep-EDV (5000:1EDV:cells), or left untreated. Cells were harvested 6h and 24h posttreatment using a cell scraper and samples were stained with DAPI(Sigma), anti-CD45 Brilliant Violet 510 (BioLegend), anti-CD86 APC-Cy7(BioLegend), and anti-CD206 AF488 (R&D Systems) and assessed by flowcytometry.

Macrophage and DC/Tumor Cell Co-Cultures:

CT26Ep12.1 and 4T1 cells were harvested with Versene (Gibco) and cellswere collected in 1 mL Eppendorf tubes. Cells were resuspended in 1 mLDMEM (Sigma) supplemented with 10% FBS (Bovogen) containing: Ep-EDV(1000:1 and 5000:1—EDV:cells); Ep-EDV-682 (500:1 and 1000:1—EDV:cells);Ep-EDV-Dox (10,000:1—EDV:cell), 100 pmol PNU-159682, 5 μM Doxorubicin,or media alone. Drug and EDV amounts were established via MTS andXCELLigence real time experiments such that chosen concentrationsresulted in the initiation of cell death within the first 24h posttreatment. Cells were then washed thoroughly with PBS to remove anynon-adherent EDV or excess drug. Treated tumor cells were culturedovernight with either RAW264.7 or BMDC at a 1:1 ratio of tumor cells:RAW264.7/BMDC/JAWS II. Supernatants were collected for ELISA analysis.RAW264.7/tumor cell co-cultures were collected using a cell scraper andsamples were stained with DAPI (Sigma), anti-CD45 Brilliant Violet 510(BioLegend), anti-CD86 APC-Cy7, and anti-CD206 AF488 and assessed byflow cytometry. JAWS II/tumor cell and BMDC/tumor cell co-cultures werecollected with versene and stained with DAPI (Sigma), CD11b AF488(Abcam), CD11c PE (Molecular Probes), anti-CD45 Brilliant Violet 510 orPECy5 (BioLegend), anti-CD86 APC-Cy7, MHC Class II PECy5 (ThermoFisher), MHC Class II Brilliant Violet 421 (BioLegend), 7-AAD(BioLegend), and/or CD80 PE (Thermo Fisher) and assessed by flowcytometry. RNA was extracted from BMDC/tumor cell co-cultures using anRNAeasy Plus Mini Kit (Qiagen) according to the manufacturer's protocol.Briefly, cells were lysed and homogenized in RLT buffer, and passedthrough a gDNA eliminator spin column. 70% ethanol was added to the flowthrough and samples were then passed through an RNeasy spin column,washed and eluted in RNase-free water. RNA concentration was determinedon an Eppendorf biophotometer plus. The RNA was used to reversetranscribe cDNA using a SuperScript™VILO™cDNA Synthesis Kit (ThermoFisher) according to the manufacturer's protocol. The transcribed cDNAwas diluted 1:2 for qPCR. Each qPCR reaction contained 5 uL TaqMan fastadvanced master mix (Thermo Fisher), 0.5 uL 20× Taqman primer/probe mix(IFN(Mm03030145_gH, IFNb1 Mm00439552_s1, GAPDH Mm99999915_g1; ThermoFisher) and 2.5 uL of water. 8 μL of the mix plus 2 μL of cDNA was addedinto 96 well plate. qPCR was performed using an Applied BiosystemsReal-Time PCR System. Data was exported to excel and the relativequantitation was calculated from the ΔΔCt.

In Vivo Tumor Models:

All animal work was performed in accordance with the EnGeneIC animalethics guidelines under AEC 1/2016, AEC 14/2016, AEC 15/2011, and AEC11/2017. For the 4T1 and CT26Ep12.1 model, female BALB/c mice wereobtained from Animal Resources Centre at 6-8 weeks of age. For T84 andA549/MDR models BALB/c Fox1^(nu)/ARC were obtained from Animal ResourcesCentre at 5-7 weeks of age.

After at least 1 week of observation, mice were injected with 5×10⁴ 4T1cells per 50 μl PBS into the 3rd mammary fat pad on the right hand sideor 2×10⁵ CT26Ep12.1 per 100 μl PBS subcutaneously into the right flankof BALB/c mice. For human xenografts, 5×10⁶ A549/MDR or 1×10⁷ T84 per100 μl PBS/Matrigel (Sigma) was subcutaneously injected into the rightflank. Treatment was commenced on day 7 post tumor induction for the 4T1model, when the average tumor size was ˜90 mm³, and on day 9 for theCT26Ep12.1 model when the average tumor size was ˜125 mm³ Mice weretreated via i.v, tail vein injection three times weekly for 2 weeks withone of the following treatments: Saline, 1×10⁹ EpCAM targeted EDVs(Ep-EDV), or 1×10⁹ EpCAM targeted EDVs loaded with PNU-159682(Ep-EDV-682). Tumors were measured 3 times/week and tumor volume wascalculated as t/6(Length×Width×Height). At the end of the 2 week period,mice were humanely euthanized and tumors and spleens collected for exvivo analysis. Treatment of A549/MDR and T84 tumors was commenced whentumors reached 100-120 mm³ and 120-150 mm³ respectively. Mice weretreated with Saline, 1×10⁹ EGFR targeted EDVs loaded with Doxorubicin(EGFR-EDV-Dox), 1×10⁹ EGFR targeted EDVs loaded with PNU-159682(EGFR-EDV-682), or 1×10⁹ non-targeted EDVs loaded with PNU-159682(EDV-682).

Isolation of CD11b⁺ Cells from 4T1 and CT26Ep12.1 Tumors:

Tumors were dissected, weighed, and enzymatically digested using aTissue Dissociation Kit (Miltenyi Biotec) at 37° C. according to themanufacturer's instructions, using the GentleMACS™ Dissociator.Following dissociation, red blood cells were removed using RBC lysisbuffer (Sigma). After washing, cells were passed through a 70 μm cellstrainer to remove any clumps. CD11b⁺ cells were purified by positiveselection using CD11b MACS beads (Miltenyi Biotec) on LS column on theMACS separator (Miltenyi Biotec). The purity of the isolated CD11b⁺ cellpopulation was assessed by flow-cytometry with an APC anti-mouse CD11b(Biolegend) and shown to be ˜80% pure (FIG. 39A).

Isolation of NK and CD8 from Spleens:

Spleens were homogenized using a Dounce homogenizer and filtered through70pM mesh strainers to obtain single cell suspension followed byerythrocyte lysis using RBC lysis buffer. Splenocytes were then washedand a cell count performed before progressing to NK or CD8+ T-cellisolation. NK cells and CD8+ T cells were isolated from dissociatedspleen cells by negative selection using either the NK Cell Isolation IIkit (Miltenyi Biotec) or the CD8a+ T Cell Isolation Kit (MiltenyiBiotec), according to the manufacturer's instructions. Cells wereseparated by using an LS column on the MACS separator (Miltenyi Biotec).NK cell and CD8+ T-cell preparations were assessed by flow-cytometry andNK cell purity was consistently greater that 90% (FIG. 39B) while CD8+T-cell purity was consistently greater than 86% (FIG. 39C). NK cellswere rested overnight in RPMI-1640 media supplemented with 10% FBS at37° C. prior to the NK cell-mediated cytolysis assay. CD8+ T-cells wereadded to tumor cells immediately following isolation to assess CD8+T-cell cytolysis.

XCELLigence Monitored CD11b+, CD8+, and NK Cell Cytolysis of TumorCells:

Cell growth characteristics and tumor cell death were monitored in realtime by the xCELLigence DP system. To do so, circular electrodescovering the base of the tissue culture wells detect changes inelectrical impedance and convert the impedance values to a Cell Index(CI). Cell Index measurements directly correspond to the strength ofcell adhesion and cell number. Target cells (4T1, CT26Ep12.1, A549/MDR,or T84) were seeded into an E-Plate 16. Cells were allowed to attach andproliferate till they had reached their logarithmic growth phase. Theeffector cells (CD11b+ cells, NK cells, or CD8+ T-cells) were added tothe target cells at the following effector-to-target cell ratios: 5:1(CD11b+: tumor cell), 20:1 (NK cell: mouse tumor cell), 10:1 (NK:humantumor cell), and 30:1 (CD8+ T-cell: tumor cell). After addition ofeffector cells, the system took regular measurements (every 5 or 15 min)for 3-4 days to monitor immune cell-mediated killing of tumor cells.

NK Cell Mediated Cytolysis Inhibition:

Mouse tumor cell lines were initially screened for NK cell ligandexpression via flow cytometry with anti-Rae-1α/β/γ-PE (Miltenyi Biotec),anti-H60a-PE (Miltenyi Biotec), and anti-MULT-1 PE (R&D Systems). For NKcell-mediated cytolysis inhibition based on these ligand expressionlevels, the effector NK cells were added to target cells in the presenceof 3 μg/ml of blocking mAb to the following NK cell ligands:anti-RAE-1αβγ (R&D Systems) or anti-H60 (R&D Systems) separately and asmixture. xCELLigence data was transformed in Excel and exported to Prism(GraphPad Software) for graphing and statistical analysis.

Tumor/Spleen Flow Cytometry:

Tumors and spleens were dissociated as described above. Following redblood cell lysis, cells were incubated with Fc block 1:10 in MACS buffer(Miltenyi Biotec) for 10 min. After the 10 min incubation, cells werewashed once and incubated with a primary antibody panel in MACS bufferfor 15 min on ice in the dark. Cells were washed 2 times and thenresuspended in MACS buffer for flow cytometric analysis. The followingantibodies were used in T-cell, NK cell, and macrophage staining panels:anti-CD45 PECy7 (BioLegend), anti-CD45 BV510 (Biolegend), anti-CD3eAPC-eFluor780 (eBioscience), anti-CD3 APC (Molecular Probes), anti-CD4PE-TR (Abcam), anti-CD8a FITC (eBioscience), anti-CD8 BV510 (BioLegend),anti-CD25 PE (Abcam), anti-CD314 (NKG2D) PE-eFluor610 (eBioscience),anti-CD335 (NKp46) PECy7 (BioLegend), anti-CD27 BV421 (BioLegend),ant-CD183 (CXCR3) BV510 (BioLegend), anti-NKG2A/C/E FITC (eBioscience),anti-CD11b APC (BD Pharmingen), anti-Ly6C FITC (BioLegend), anti-Ly6GBV510 (BioLegend), anti-F4/80 PE Dazzle594 (BioLegend), anti-CD206 PECy7(BioLegend), and anti-CD86 APC-Cy7 (BioLegend). Single stained controlsand/or versacomp (Beckman Coulter) beads were used for fluorescencecompensation. DAPI (Sigma), propidium iodide (Sigma), DRAQ5 (ThermoFisher), or Live/Dead Yellow (Thermo Fisher) were used for live celldetection. Unstained and isotype controls were employed to determineauto-fluorescence levels and confirm antibody specificity.

Cytokine and Chemokine Detection (Tumor and Splenocytes):

To measure the interstitial cytokine and chemokine levels in the mousetumors, tumors were carefully dissected removing all skin, placed intoserum free media, and weighed. Tumors were then gently broken up usingEppendorf micropestles (Sigma), ensuring no large pieces were visible.The cell suspension was centrifuged and the supernatant collected andstored at −80° C. until analysis. For splenocyte/tumor cell co-cultures,spleens were dissociated and tumors were enzymatically digested aspreviously described. Splenocytes and tumors from the same mouse werethen placed into tissue culture plates at a ratio of 10:1(Splenocytes:tumor cells) and cultured for up to 72 h. Supernatant wascollected at 24, 48 and 72 h and stored at −80° C. until analysis. Tumorand splenocyte supernatant was analyzed for mouse IL-1β, TNF-α, IL-2,IL-4, IL-6, IFNα, IFNγ, RANTES and MIP-1α according the manufacturer'sinstructions. The IFNα kit was obtained from PBL Assay Science, whileIL-1β, TNF-α, IL-2, IL-4, IL-6, IFNγ, RANTES (CCL5) and MIP-1α (CCL3)duoset kits were obtained from R&D systems. Each ELISA was developedusing the 3,3′,5,5′-tetramethylbenzidine (Sigma) substrate. Microwellplates were read in a Biotek uQuant plate reader at 450 nm with 540 nmas the reference wavelength. KC junior software was used to fit 4parameter logistic curves to the standards and interpolate the samples.The minimum detectable concentration (MDC) of each assay was calculatedby multiplying the s.d. of the response by 10 and dividing by the slopeof the standard curve at the inflection point.

Confocal Microscopy:

4T1 cells were seeded on Lab-Tek chamber slides (Sigma) and left toattach and grow for 24 h. Isolated CD8⁺ T-cells were added to the 4T1cells and left for 8 h, at which time, cells were fixed in 4%paraformaldehyde. Cells were washed and permeabilized with 0.5%triton-x-100 in PBS (PBST). Cells were blocked with 3% BSA for 30 minfollowed by incubation with the primary anti-perforin antibody (Abcam)diluted in PBST. After washing, cells were incubated with the secondarygoat anti-rat IgG Alexafluor 488 (Abcam), followed by incubation withAlexaFluor 568 Phalloidin (Thermo Fisher). Cells were mounted withProlong Diamond Antifade with DAPI (Thermo Fisher) and sealed with nailpolish prior to imaging. Images were acquired on a Zeiss LSM 780, andimages were merged and processed in Image J.

Case Presentation of Stage IV Pancreatic Ductal Adenocarcinoma:

A 67-year old Caucasian woman, CEB, whose primary symptom was jaundice,had previously undergone a complete Whipple procedure for pancreaticductal adenocarcinoma (PDAC), to remove the pancreas, the gallbladder,the duodenum, the spleen and a portion of the stomach and surroundinglymph nodes. She had tumours in both the head and tail of the pancreasand her disease was diagnosed as Stage IV. She was treated withgemcitabine followed by FOLFIRINOX at another institution but haddeveloped extensive metastatic disease in the liver on treatment. At theconclusion of her chemotherapy, sixteen months after her Whippleprocedure, she had exhausted all treatment options, her weight was downfrom 62 kg to 45 kg, and she sought experimental EDV treatment whichcould be administered under the Australian Therapeutic GoodsAdministration (TGA) compassionate use scheme, and had been previouslytested in a Phase I trial for mesothelioma (van Zandwijk et al., 2017)and recurrent glioblastoma (Whittle et al., 2015). CEB was dosed twiceweekly for 7 weeks in her first cycle in the oncology ward at RoyalNorth Shore Hospital, Sydney. However, because of her weakened state,and to potentially build tolerance to the lipopolysaccharide inherent inthe EDV, doses were slowly escalated within the cycle (Table 17, below).

TABLE 17 Week Dose EDV concentration 1 1, 2 0.75 × 10⁸  2 3, 4 1.0 × 10⁹3 5, 6 1.25 × 10⁹  4 7, 8 1.5 × 10⁹ 5  9, 10 2.0 × 10⁹ 6 11, 12 2.0 ×10⁹ 7 13, 14 2.5 × 10⁹

The dosed was administered over 20 min via a 20 ml niki pump andpremedications were given prior to dosing as before (van Zandwijk etal., 2017). Serum biochemistry, haematology and cytokine expression wasevaluated pre and 3 hours post each dose. CA19-9 and C-reactive proteinlevels were monitored at least bi-weekly. Peripheral Blood mononuclearcells (PBMCs) were examined by flow cytometry prior to dosing and at theend of the cycle for changes in anti-tumour immune cell numbers. Tumourtissue was obtained from the original surgical resection and PDAC cellswere cultured and tested for drug sensitivity and surface receptorsexpression.

Statistics:

All statistical analysis was performed using the GraphPad Prism softwarepackage. Data is represented as mean±standard deviation (SD) or standarderror of the mean (SEM). Statistical significance between 2 groups wasdetermined by a student's t-test. Statistical significance betweengroups of 3 or more was determined by a one way ANOVA, followed by theTukey's multiple comparison test. Significance for tumor regressionstudies was determined by a two way ANOVA followed by the Tukey'smultiple comparison test. For all tests, p values were as follows: *p≤0.05, ** p≤0.01, *** p≤0.001, and **** p≤0.0001.

Example 22: EDV_(αGC) Treatment of JAWSII Cells and the SubsequentSurface Presentation of αGC Through CD1d Ligand

This example contrasts EDV-delivery of αGC and free αGC against cancercells.

Cells used: Mouse immature monocytes JAWSII (ATCC® CRL-11904™).

Preparation Perfecta3D 96-Well Hanging Drop Plate:

The upper and lower side tray reservoirs of the 3D hanging drop plateswere filled with melted 1% agarose using a P1000 pipette (1 g agarosedissolve in 100 ml of water, dissolved in microwave and allowed to coolto ˜50° C.). The plates were allowed to dry and settle at roomtemperature for at least 30 min. The outside wells of the hanging dropplate were then filled with 50 μl of sterile cell culture media (withoutcells)/well.

Treatment of JAWSII Spheroids with EDV_(αGC):

JAWSII cells were treated with 1000 ng/ml αGC (positive control); emptyminicells and minicells_(αGC) compared to untreated cells and collectedat 8h, 16h, 24h and 48h post-treatment (FIG. 46A-46D).

Dissociation of JAWSII Cells into Single-Cell Suspensions:

JAWSII cells were grown as semi-suspension cultures in T25 or T75flasks. The culture media was carefully collected into a sterile 50 mltube by pipetting using a pipette-aid and the culture surface of theflask was washed 2× with 5 ml of sterile PBS, and collected in the samesterile 50 ml tube after each wash. The adherent cells were collected bythe addition of 5 ml of 0.25% trypsion/EDTA and incubated at 37° C. for3 min or until all the cells were lifted from the surface of the flask.The lifted cells were carefully broken up into single cells by gentlepipetting using a pipette-aid and transferred into the sample sterile 50ml tube used in previous steps. The cell suspension was then centrifugedat 300 g for 7 min and the supernatant was carefully decanted. The cellpellet was dissociated by flicking the bottom of the tube with a fingerand resuspended in 5 ml of pre-warmed JAWSII culture media. The cellsuspension was further dissociated into single cells by carefulpipetting using a pipette-aid. To determine the cell number, 10 μl ofthe cell suspension was mixed with 10l of trypan blue solution andanalysed using an EVE automated cell counter.

Initial Treatment Preparation:

6 hanging drop suspension samples were used for each treatment group pertime point. 5×10⁴ JAWSII cells and 5×10⁸ minicells (1:1000 minicell tocell ratio) were used for each treatment sample and cultured in JAWSIIcell culture media in a total volume of 50 μl. Extra untreated sampleswere prepared for isotype controls. The appropriate amount of minicellswere pelleted by centrifugation at 12,000 g for 7 min and thesupernatant was carefully removed by pipetting. Appropriate amount oflive JAWS cells (based on the cell count from the previous section) wereadded to the pelleted minicells. The minicells were then dissociatedinto single-minicells-cell suspensions by gentle pipetting. The finalvolume of each sample was then made up by the addition of sterileculture media. For the untreated and αGC treated samples, 5×10⁴ JAWSIIcells were used for each sample and cultured in JAWSII cell culturemedia in a total volume of 50 μl. Appropriate amount of live JAWSIIcells were transferred into Eppendorf tubes. The final volume of eachsample was then made up by the addition of sterile culture media. 1000ng/mL of αGC was added directly into the cell suspension for the JAWSIIcells treated with 1000 ng/ml αGC (positive control) treatment group.The samples were then carefully seeded into each well of the hangingdrop plates at 50 μl of treatment suspension/well and incubated at 37°C. at 5% CO₂ until collection.

Staining the Treated JAWSII Cells with Anti-Alpha GalCer:mCD1d ComplexMonoclonal Antibody:

The entire content of each hanging drop well was carefully collectedusing a P200 pipette and transferred into an Eppendorf tube. A total of6 samples were collected for each treatment group into 1 tube. 1:1000 PEconjugated anti-mouse alpha GalCer:mCD1d complex monoclonal antibody and1:1000 PE conjugated mouse IgG1 isotype control were added intoappropriate samples and mixed by gentle vortexing. GalCer:mCD1dmonoclonal antibody binds to the cell surface exposed portion of theGalCer:CD1d complex. The samples were then incubated at room temperaturefor 20 min in the dark. Samples were then pelleted by centrifugation at350 g for 5 min. The supernatant was removed by careful pipetting andthe pellets were re-suspended and washed once in 500 μL FACS buffer. Thecells were then collected by centrifugation at 350 g for 5 min,resuspended in 250 μL FACS buffer and transferred into FACS tubes. 1 μLof DAPI was added into each sample and mixed by gently swirling of thetubes. The samples were then analyzed using a Gallios flow cytometer.

Results:

Flow cytometry data (FIGS. 45A-45E) showed a clear shift after stainingwith anti-GalCer:mCD1d for JAWSII cells treated with minicells_(α-GC)and with free α-GC compared to JAWSII cells treated with minicells aloneand untreated. This positive staining, confirms the successful deliveryof α-GC by minicells to JAWSII cells and subsequent antigen presentationon the cell surface by the CD1d molecule which presents glycolipids onthe cell surface. Presentation of α-GC is a crucial step which leads toreceptor recognition by invariant NKT cells triggering off a type II IFNcascade essential in anti-tumor activity.

Example 23: In Vivo Studies Using Combination Treatment of^(Ep)Minicell_(Dox) and Minicell_(α-GC) in a Syngeneic Mouse Model(^(Ep)CT26 Murine Colon Cancer in Balb/c Mice)

This example illustrates the efficacy of minicell contained therapeuticand minicell contained interferon type II agonist against tumors. Thisresult demonstrates that compositions lacking interferon type I agonistscan be used to effectively treat tumors.

Mice and Treatments (Experiments 1-3):

Balb/c mice, female, 6-7 weeks old were obtained from the AnimalResources Company in Western Australia. The mice were acclimatized forone week before the experiments commenced. CT26 cells (mouse coloncancer) were stably transformed with a plasmid expressing EpCAM antigenand a stable clone (Epclone 12.1) was established. This clone expressedEpCAM on the surface of the cells. All animal experiments were performedin compliance with National Health and Medical Research Council,Australia guidelines for the care and use of laboratory animals, andwith EnGeneIC Animal Ethics Committee approval.

CT26 (Epclone 12. 1) isografts were established by injecting 2×10⁵ cellsper 100 l PBS subcutaneously on the left flank of each mouse. The tumorsgrew to the ˜125 mm³ starting volume within 8 days post implantation.The mice were randomly distributed into groups with 8 mice for eachtreatment group. Tumors were treated with ^(Ep)minicell_(Dox),minicell_(α-GC) and ^(Ep)minicell_(Dox)+ minicell_(α-GC) (combination)compared to saline treatment alone.

Dosing was carried out 3× per week for 2 weeks. ^(EP)minicell_(Dox) wasdosed at 1×10⁹ minicells per dose in single and in combinationtreatments. minicell_(α-GC) was dosed at 1×10⁷ in experiments 1 (FIG.40) and 3 (FIG. 42) and 1×10⁸ in experiment 2 (FIG. 40); where thesaline group was also challenged when the tumor volume reached 800 mm³.

Results:

All 3 experiments showed a marked halt in tumor progression forcombination treatment groups receiving ^(Ep)minicell_(Dox)+minicell_(α-GC) compared to saline and ^(Ep)minicell_(Dox) treatments.This result supports the theory of an immune adjuvant effect by theaddition of minicell_(α-GC) treatment to ^(Ep)minicell_(Dox). Treatmentwith minicell_(α-GC) alone also showed a halt in tumor progression forall 3 experiments, though not to the extent of the combinationtreatment, as best seen in experiment 2.

In experiment 2, saline treated control tumors demonstrated dramatictumor regression following a treatment change to drug and α-GC EDVmediated combination therapy (FIG. 41). Tumours that had reached 800 mm³dropped to below 600 mm³ in 3 days before the experiment was terminated.

Dose Evaluation of Different Sized Tumors; Mice and Treatments(Experiment 4):

CT26 (Ep clone 12.1) isograft was established by injectingsubcutaneously 2×10⁵ cells/100 l PBS into the left flank of female, 6-7weeks old Balb/c mice. The tumours were grown to ˜200-250 mm³ or 600-800mm³ before treatments commenced. The mice were randomised into 6 groups,3 mice per group. Mice received one dose only. Treatment groupsincluded; Saline (FIG. 43C), ^(EP)minicell_(D)ox (1×10⁹) (FIG. 43F),minicell_(α-GC) (1×10⁶) (FIG. 43E), minicell_(α-GC) (1×10⁷) (FIG. 43D),^(EP)minicell_(Dox) 1×10⁹+ minicell_(α-GC) (1×10⁶) (FIG. 43B),^(EP)minicell_(Dox) (1×10⁹)+ minicell_(α-GC) (1×10⁷) (FIGS. 43A-43F).

Mice were sacrificed at 24 hrs post treatment for 200-250 mm³ (FIG. 43)tumors and at 16 hrs and 24 hrs for 600-800 mm³ tumors (FIGS. 44A-44B).

Results:

The effect of minicell_(α-GC) dosing, alone and in combination, in CT26syngeneic tumor bearing Balb/c mice was further investigated by treatingdifferent sized tumors with a single dose as described above.Interestingly it was found that in both, mice carrying tumors of 200-250mm³ as well as 400-600 mm³, the tumours developed a marked necrosis(black color) within 24 hours of dosing. This effect was more pronouncedin the larger tumours and not seen in the control groups.

In sum, this data shows that a combination of minicell containedtherapeutic and a interferon type II agonist against demonstratesefficacy against tumors. This result demonstrates that compositionslacking interferon type I agonists can be used to effectively treattumors

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the methods and compositionsof the present invention without departing from the spirit or scope ofthe invention. Thus, it is intended that the present invention cover themodifications and variations of this invention, provided they comewithin the scope of the appended claims and their equivalents.

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What is claimed is:
 1. A composition comprising: (a) a therapeuticallyeffective dose of purified, intact bacterially derived minicellscomprising at least one anti-neoplastic agent, wherein theanti-neoplastic agent comprises nemorubicin, PNU-159682, idarubicin,daunorubicin, caminomycin, and/or and (b) an interferon type I agonist,an interferon type II agonist, or a combination of an interferon type Iagonist and an interferon type II agonist, wherein the interferon type Iagonist is an oligonucleotide, wherein the oligonucleotide is doublestranded DNA or DNA-RNA hybrids and comprises a sequence of at leastabout 40 nucleotides, and wherein the interferon type II agonist isselected from the group consisting of C-glycosidific form ofα-galactosylceramide (α-C-GalCer), α-galactosylceramide (α-GalCer), 12carbon acyl form of galactosylceramide (β-GalCer),β-D-glucopyranosylceramide (β-GlcCer),1,2-Diacyl-3-0-galactosyl-sn-glycerol (BbGL-II), diacylglycerolcontaining glycolipids (Glc-DAG-s2), ganglioside (GD3),gangliotriaosylceramide (Gg3Cer), glycosylphosphatidylinositol (GPI),α-glucuronosylceramide (GSL-1 or GSL-4), isoglobotrihexosylceramide(iGb3), lipophosphoglycan(LPG), lyosphosphatidylcholine (LPC),a-galactosylceramide analog (OCH), threitolceramide, and a combinationthereof.
 2. The composition of claim 1, wherein element (b) of thecomposition comprises: (i) a therapeutically effective dose of purified,intact bacterially derived minicells comprising an interferon type Iagonist; or (ii) a therapeutically effective dose of purified, intactbacterially derived minicells comprising an interferon type II agonist;or (iii) a combination of: (1) a therapeutically effective dose ofpurified, intact bacterially derived minicells comprising an interferontype I agonist; and (2) a therapeutically effective dose of purified,intact bacterially derived minicells comprising an interferon type IIagonist.
 3. The composition of claim 1, wherein: (a) the anti-neoplasticagent and the interferon type I agonist, the interferon type II agonist,or the combination of an interferon type I agonist and an interferontype II agonist, are packaged within two or more purified, intactbacterially derived minicells; or (b) the anti-neoplastic agent and theinterferon type I agonist, the interferon type II agonist, or thecombination of an interferon type I agonist and an interferon type IIagonist are packaged within three separate populations of purified,intact bacterially derived minicells.
 4. The composition of claim 1comprising the anti-neoplastic agent, the interferon type I agonist, andthe interferon type II agonist, wherein: (a) the anti-neoplastic agent,the interferon type I agonist, and the interferon type II agonist arecomprised within the same minicell; (b) the anti-neoplastic agent andthe interferon type I agonist are comprised within a first minicell, andthe interferon type II agonist is comprised within a second minicell;(c) the anti-neoplastic agent and the interferon type II agonist arecomprised within a first minicell, and the interferon type I agonist iscomprised within a second minicell; (d) the anti-neoplastic agent iscomprised within a first minicell, and the interferon type I agonist andthe interferon type II agonist are comprised within a second minicell;or (e) the anti-neoplastic agent is comprised within a first minicell,the interferon type I agonist is comprised within a second minicell, andthe interferon type II agonist is comprised within a third minicell. 5.The composition of claim 1, wherein the composition does not comprise aninterferon type I agonist.
 6. The composition of claim 1, wherein: theanti-neoplastic agent is PNU-159682.
 7. The composition of claim 1,wherein: (a) the oligonucleotide comprises a sequence of at least about50 nucleotides or at least about 60 nucleotides.
 8. The composition ofclaim 1, wherein the interferon type I agonist is an oligonucleotideselected from the group consisting of double stranded Z-DNA and B-DNA.9. The composition of claim 1, wherein: the interferon type II agonistis α-galactosylceramide (α-GalCer).
 10. The composition of claim 1,further comprising: (a) a bispecific ligand bound to the minicellscomprising the anti-neoplastic agent; and/or (b) a bispecific ligandbound to the minicells comprising the type I interferon agonist; and/or(c) a bispecific ligand bound to the minicells comprising the type IIinterferon agonist.
 11. The composition according to claim 10, whereinthe bispecific ligand: (a) comprises a first arm that carriesspecificity for a minicell surface structure and a second arm thatcarries specificity for a non-phagocytotic mammalian cell surfacereceptor; and/or (b) comprises a first arm that carries specificity fora minicell surface structure and a second arm that carries specificityfor a non-phagocytotic mammalian cell surface receptor and wherein theminicell surface structure is an O-polysaccharide component of alipopolysaccharide on the minicell surface; and/or (c) comprises a firstarm that carries specificity for a minicell surface structure and asecond arm that carries specificity for a non-phagocytotic mammaliancell surface receptor wherein the non-phagocytotic mammalian cellsurface receptor is capable of activating receptor-mediated endocytosisof the minicell; and/or (d) comprises a bispecific antibody or antibodyfragment; and/or (e) comprises a bispecific antibody or antibodyfragment and wherein the antibody or antibody fragment comprises a firstmultivalent arm that carries specificity for a bacterially derivedminicell surface structure and a second multivalent arm that carriesspecificity for a cancer cell surface receptor, wherein the cancer cellsurface receptor is capable of activating receptor-mediated endocytosisof the minicell.
 12. The composition of claim 1, wherein the compositioncomprises fewer than about 1 contaminating parent bacterial cell per10⁷minicells, fewer than about 1 contaminating parent bacterial cell per10⁸minicells, fewer than about 1 contaminating parent bacterial cell per10⁹minicells, fewer than about 1 contaminating parent bacterial cell per10¹⁰minicells, or fewer than about 1 contaminating parent bacterial cellper 10¹¹minicells.
 13. The composition of claim 1, further comprising apharmaceutically acceptable carrier.
 14. The composition of claim 1,wherein the minicells are approximately 400 nm in diameter.
 15. Thecomposition of claim 1, wherein the composition is free of parentbacterial cell contamination removable through 200 nm filtration. 16.The composition of claim 1, wherein the composition comprises thefollowing amount of minicells or killed bacterial cells: (a) at leastabout 10⁹; (b) at least about 1×10⁹; (c) at least about 2×10⁹; (d) atleast about 5×10⁹; (e) at least 8×10⁹; (f) no more than about 10¹¹; (g)no more than about 1×10¹¹; (h) no more than about 9×10¹⁰; or (i) no morethan about 8×10¹⁰.
 17. The composition of claim 1, comprising theanti-neoplastic agent, the interferon type I agonist, and interferongamma.